Bcma antibodies and use of same to treat cancer and immunological disorders

ABSTRACT

The invention provides humanized antibodies that specifically bind to BCMA. The antibodies are useful for treatment and diagnoses of various cancers and immune disorders as well as detecting BCMA.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of U.S. application Ser. No. 16/411,867filed May 14, 2019, which is a continuation of U.S. application Ser. No.15/434,921 filed Feb. 16, 2017, now abandoned, which claims the benefitof U.S. provisional application no. U.S. 62/296,594 filed Feb. 17, 2016,and U.S. provisional application No. 62/396,084 filed Sep. 16, 2016, allof which are incorporated herein by reference in their entirety for allpurposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named0269_Sequence_Listing.txt created on May 9, 2019 and containing 73 KB,which is hereby incorporated by reference.

BACKGROUND

B-cell maturation antigen (BCMA, CD269) is a member of the TNF receptorsuperfamily. Expression of BCMA is restricted to the B-cell lineagewhere it is predominantly expressed in the interfollicular region ofgerminal centers and on differentiated plasma cells and plasma blasts.BCMA binds to two distinct ligands, a proliferation inducing ligand(APRIL) and B-cell activating factor (BAFF, also known as BlyS, TALL-1,and THANK). The ligands for BCMA bind two additional TNF receptors,transmembrane activator and calcium modulator and cyclophilin ligandinteractor (TACI) and BAFF receptor (BAFF-R also called BR3). TACI bindsAPRIL and BAFF, whereas BAFF-R shows restricted but high-affinitybinding to BAFF. Together, BCMA, TACI, BAFF-R, and their correspondingligands regulate different aspects of humoral immunity, B-celldevelopment, and homeostasis.

BCMA is virtually absent on naïve and memory B cells (Novak et al.,Blood 103, 689-94 (2004)) but it is selectively induced during plasmacell differentiation where it may support humoral immunity by promotingthe survival of normal plasma cells and plasma blasts (O'Conner et al.,J. Exp Med. 199, 91-98 (2004)). BCMA has been reported to be expressedin primary multiple myeloma (MM) samples. BCMA has also been detected onthe Reed-Sternberg cells (CD30⁺) from patients with Hodgkin's disease.It has been reported based on knockdown experiments that that BCMAcontributed to both proliferation and survival of a Hodgkin's diseasecell line (Chiu et al., Blood 109, 729-39 (2007)).

SUMMARY OF THE CLAIMED INVENTION

The invention provides a humanized, chimeric or veneered antibody, whichis a humanized or chimeric form of an antibody deposited as ATCCPTC-6937. Optionally the antibody comprises a mature heavy chainvariable region having at least 90% sequence identity to hSG16.17 VH3(SEQ ID NO: 13) and a mature light chain variable region having at least90% sequence identity to hSG16.17 VK2 (SEQ ID NO: 19). Optionally, theantibody comprises a mature heavy chain variable region having at least95% sequence identity to hSG16.17 VH3 (SEQ ID NO: 13) and a mature lightchain variable region having at least 95% sequence identity to hSG16.17VK2 (SEQ ID NO: 19). Optionally, the antibody comprising the three KabatCDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three KabatCDRs (SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19) provided thatposition H58 can be occupied by N or K, position H60 can be occupied byA or N, position H61 can be occupied by Q or E, position H62 can beoccupied by K or N, position H64 can be occupied by Q or K, position H65can be occupied by G or T, position L24 can be occupied by R or L andposition L53 can be occupied by S or R. Optionally, the antibodycomprises the three Kabat CDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQID NO: 13) and three Kabat CDRs (SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQID NO: 19). Optionally, positions H58, H60, H61, H62, H64 and H65 areoccupied by N, A, Q K, Q and G respectively and L24 and L53 are occupiedby R and S respectively. Optionally, positions H20, H48, H69, H71, H73,H76, H80, H88, H91 and H93 are occupied by L, I, M, A, K, N, V, A, F,and T respectively, and positions L46, L48 and L87 are occupied by V, Vand F respectively. Optionally, the mature heavy chain variable has thesequence of hSG16.17 VH3 (SEQ ID NO: 13) and the mature light chainvariable region has the sequence of hSG16.17 VK2 (SEQ ID NO: 19).

The invention further provides a humanized, chimeric or veneeredantibody, which is a humanized, chimeric or veneered form of the ratSG16.45 antibody having the VH (SEQ ID NO: 23) and VK (SEQ ID NO: 33)sequences. Optionally, the antibody comprises a heavy chain maturevariable region having at least 90% sequence identity to hSG16.45 VH5(SEQ ID NO: 31) and a mature light chain variable region having at least90% sequence identity to hSG16.45 VK2 (SEQ ID NO: 36). Optionally, theantibody comprises a mature heavy chain variable region having at least95% sequence identity to hSG16.45 VH5 (SEQ ID NO: 31) and a mature lightchain variable region having at least 95% sequence identity to hSG16.45VK2 (SEQ ID NO: 36). Optionally, the comprises the three Kabat CDRs (SEQID NOs: 152-154) of hSG16.45 VH5 (SEQ ID NO: 31) and three Kabat CDRs(SEQ ID NOs: 179-181) of hSG16.45 VK2 (SEQ ID NO: 36) provided thatpositions H50 can be occupied by A or S and position L24 can be occupiedby R or L and position L26 can be occupied by S or T. Optionally, theantibody comprises the three Kabat CDRs (SEQ ID NOs: 152-154) ofhSG16.45 VH5 (SEQ ID NO: 31) and three Kabat CDRs (SEQ ID NOs: 179-181)of hSG16.45 VK2 (SEQ ID NO: 36). Optionally positions H30, H93 and H94are occupied by N, T and S respectively. Optionally, the mature heavychain variable region has the sequence of hSG16.45 VH5 (SEQ ID NO: 31)and the mature light chain variable region has the sequence of hSG16.45VK2 (SEQ ID NO: 36) or the mature heavy chain variable region has thesequence of hSG16.45 VH1 (SEQ ID NO: 27) and the mature light chainvariable region has the sequence of hSG16.45 VK1 (SEQ ID NO: 35) or themature heavy chain variable region has the sequence of hSG16.45 VH1 (SEQID NO: 27) and the mature light chain variable region has the sequenceof hSG16.45 VK3 (SEQ ID NO: 37).

In any of the above antibodies, the mature heavy chain variable regioncan be fused to a heavy chain constant region and the mature light chainvariable region can be fused to a light chain constant region.Optionally, the heavy chain constant region is a mutant form of naturalhuman constant region which has reduced binding to an Fcγ receptorrelative to the natural human constant region. Optionally, the heavychain constant region is of IgG1 isotype. Optionally, the heavy chainconstant region has an amino acid sequence comprising SEQ ID NO: 5 andthe light chain constant region has an amino acid sequence comprisingSEQ ID NO: 3. Optionally, the heavy chain constant region has an aminoacid sequence comprising SEQ ID NO:7 (S239C) and the light chainconstant region has an amino acid sequence comprising SEQ ID NO:3.Optionally, the antibody is a naked antibody. Optionally, the antibodyis conjugated to a cytotoxic or cytostatic agent. Optionally, theantibody is conjugated to a cytotoxic agent. Optionally, the cytotoxicagent is conjugated to the via an enzyme cleavable linker. Optionally,the cytotoxic agent is a DNA minor groove binder, e.g., the cytotoxicagent having the formula

Optionally, the cytotoxic agent is MMAE or MMAF.

The invention further provides pharmaceutical compositions comprisingany of the antibodies described above and a pharmaceutically acceptablecarrier.

In one embodiment, the invention provides an antibody comprising thethree Kabat CDRs (SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) andthree Kabat CDRs (SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19). Ina further embodiment, the invention provides an antibody having a matureheavy chain variable with the sequence of hSG16.17 VH3 (SEQ ID NO: 13)and a mature light chain variable region with the sequence of hSG16.17VK2 (SEQ ID NO: 19). In another embodiment, the mature heavy chainvariable region is fused to a heavy chain constant region and the maturelight chain variable region is fused to a light chain constant region.The antibody can be, e.g., an IgG1 antibody. In another embodiment, theantibody lacks core fucosylation by fucose or a fucose analogue. Theantibodies can by formulated into a pharmaceutical composition, e.g.,with addition of a pharmaceutically acceptable carrier.

In a further embodiment, the pharmaceutical composition has a pluralityof antibodies having a mature heavy chain variable with the sequence ofhSG16.17 VH3 (SEQ ID NO: 13) and a mature light chain variable regionwith the sequence of hSG16.17 VK2 (SEQ ID NO: 19). The variable regionsof these antibodies are preferably fused to appropriate heavy and lightchain constant regions. In another embodiment the antibodies are IgG1antibodies. In a further embodiment, the plurality of antibodies hasless than about 5% of the antibodies have core fucosylation by fucose ora fucose analogue. In a further embodiment, the plurality of antibodieshas less than about 10% of the antibodies have core fucosylation byfucose or a fucose analogue. In another embodiment, the plurality ofantibodies includes about 2% antibodies with core fucosylation by fucoseor a fucose analogue. In another embodiment, the plurality of antibodiesincludes 2% antibodies with core fucosylation by fucose or a fucoseanalogue.

The invention further provides a method of treating a patient having orat risk of having a cancer that expresses BCMA comprising administeringto the patient an effective regime of an antibody as described above.Optionally the cancer is a hematological cancer. Optionally, thehematological cancer is a myeloma, leukemia or a lymphoma. Optionally,the hematological cancer is multiple myeloma. Optionally thehematological cancer is non-Hodgkin's lymphoma (NHL) or Hodgkin'slymphoma. Optionally, the hematological cancer is myelodysplasticsyndromes (MDS), myeloproliferative syndromes (MPS), Waldenström'smacroglobulinemia or Burkett's lymphoma.

The invention further provides a method of treating a patient having orat risk of having an immune disorder mediated by immune cells expressingBCMA comprising administering to the patient an effective regime of anyof the above described antibodies. Optionally, the disorder is a B cellmediated disorder. Optionally, the immune disorder is rheumatoidarthritis, systemic lupus E (SLE), Type I diabetes, asthma, atopicdermitus, allergic rhinitis, thrombocytopenic purpura, multiplesclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, and graft versus host disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the structure of BCMA.

FIG. 1B shows the structural interaction of the extracellular domain ofBCMA with BAFF.

FIG. 2 shows an antibody selection procedure.

FIG. 3 shows cell binding data and ligand blockade activity for unclonedhybridoma wells.

FIG. 4 shows blocking activity/percent inhibition of of anti-BCMAantibodies.

FIG. 5 shows inhibition of APRIL blocking titrated with anti-BCMAantibodies.

FIG. 6 shows a titration of BAFF blocking using anti-BCMA antibodies.

FIG. 7 shows alignment of hSG16.17 heavy chain variants with human VHacceptor sequence, HV1-2/HJ3. It shows rat SG16.17 vH (SEQ ID NO: 8)with Kabat CDRs (SEQ ID Nos: 39-41) and IMGT CDRs (SEQ ID NOs: 42 and43); Hu HV1-2/HJ3 (SEQ ID NO: 9) with Kabat CDRs (SEQ ID NOs: 44 and 45)and IMGT CDRs (SEQ ID NO: 46 and “AR”); hSG16.17 vH1 (SEQ ID NO: 11)with Kabat CDRs (SEQ ID NOs: 50-52) and IMGT CDRs (SEQ ID NOs: 53 and54); hSG16.17 vH2 (SEQ ID NO: 12) with Kabat CDRs (SEQ ID NOs: 55-57)and IMGT CDRs (SEQ ID NOs: 58 and 59); hSG16.17 vH3 (SEQ ID NO: 13) withKabat CDRs (SEQ ID NOs: 60-62) and IMGT CDRs (SEQ ID NOs: 63 and 64);and hSG16.17 vH4 (SEQ ID NO: 14) with Kabat CDRs (SEQ ID NOs: 65-67) andIMGT CDRs (SEQ ID NOs: 68 and 69).

FIG. 8 shows alignment of hSG16.17 heavy chain variants with human VHacceptor sequence; HV1-46/HJ3. It shows the sequences of rat SG16.17 vH(SEQ ID NO: 8) with Kabat CDRs (SEQ ID NOs: 39-41) and IMGT CDRs (SEQ IDNOs: 42 and 43); Hu HV1-46/HJ3 (SEQ ID NO: 10) with Kabat CDRs (SEQ IDNOs: 47 and 48) and IMGT CDRs (SEQ ID NO: 49 and “AR”); hSG16.17 vH5(SEQ ID NO: 15) with Kabat CDRs (SEQ ID NOs: 70-72) and IMGT CDRs (SEQID NOs: 73 and 74); and hSG16.17 vH6 (SEQ ID NO: 16) with Kabat CDRs(SEQ ID NOs: 75-77) and IMGT CDRs (SEQ ID NOs: 78 and 79).

FIG. 9 shows alignment of hSG16.17 heavy chain variants. It shows thesequences of hSG16.17 vH1-6 (SEQ ID NOs: 11-16).

FIG. 10 shows alignment of hSG16.17 light chain variants with human VKacceptor sequence; KV1-12/KJ5. It shows the sequences of rat SG16.17 vK(SEQ ID NO: 17) with Kabat CDRs (SEQ ID NOs: 80-82) and IMGT CDRs (SEQID NO: 83, “TTS”, and SEQ ID NO: 84, respectively); Hu KV1-12/KJ5 (SEQID NO: 18) with Kabat CDRs (SEQ ID NOs: 85-87) and IMGT CDRs (SEQ ID NO:88, “AAS”, and SEQ ID NO: 89, respectively); hSG16.17 vK2 (SEQ ID NO:19) with Kabat CDRs (SEQ ID NOs: 90-92) and IMGT CDRs (SEQ ID NO: 93,“TTS”, and SEQ ID NO: 94, respectively); hSG16.17 vK3 (SEQ ID NO: 20)with Kabat CDRs (SEQ ID NOs: 95-97) and IMGT CDRs (SEQ ID NO: 98, “TTS”,and SEQ ID NO: 99, respectively); hSG16.17 vK4 (SEQ ID NO: 21) withKabat CDRs (SEQ ID NOs. 100-102) and IMGT CDRs (SEQ ID NO: 103, “TTS”,and SEQ ID NO: 104, respectively); and hSG16.17 vK5 (SEQ ID NO: 22) withKabat CDRs (SEQ ID NOs: 105-107) and IMGT CDRs (SEQ ID NO: 108, “TTS”,and SEQ ID NO: 109, respectively).

FIG. 11 shows alignment of hSG16.17 light chain variants. It shows thesequences of hSG16.17 vK2, vK3, vK4, vK5 (SEQ ID NOs: 19-22).

FIG. 12 shows competition binding assay showing binding of chimericSG16.17 to human FcRIIIa.

FIG. 13: shows chimeric SG16.17 induces signal lying through FcγRIIIA.

FIG. 14 shows alignment of hSG16.45 heavy chain variants with human HVacceptor sequence HV3-23/HJ3. It shows the sequences of Rat SG16.45 vH(SEQ ID NO: 23) with Kabat CDRs (SEQ ID NOs: 110-112) and IMGT CDRs (SEQID NOs: 113-115); Hu HV3-23/HJ3 (SEQ ID NO: 24) with Kabat CDRs (SEQ IDNOs: 116 and 117) and IMGT CDRs (SEQ ID NOs: 118 and 119, and “AK”,respectively); hSG16.45 vH1 (SEQ ID NO: 27) with Kabat CDRs (SEQ ID NOs:128-130) and IMGT CDRs (SEQ ID NOs: 131-133); hSG16.45 vH2 (SEQ ID NO:28) with Kabat CDRs (SEQ ID NOs: 134-136) and IMGT CDRs (SEQ ID NOs:137-139); hSG16.45 vH3 (SEQ ID NO: 29) with Kabat CDRs (SEQ ID NOs:140-142) and IMGT CDRs (SEQ ID NOs: 143-145); and hSG16.45 vH4 (SEQ IDNO: 30) with Kabat CDRs (SEQ ID NOs: 146-148) and IMGT CDRs (SEQ ID NOs:149-151).

FIG. 15 shows alignment of hSG16.45 heavy chain variants with human HVacceptor sequence HV3-74/HJ3. It shows the sequences of Rat SG16.45 vH(SEQ ID NO: 23) with Kabat CDRs (SEQ ID NOs: 110-112) and IMGT CDRs (SEQID NOs: 113-115); Hu HV3-74/HJ3 (SEQ ID NO: 25) with Kabat CDRs (SEQ IDNOs: 120 and 121) and IMGT CDRs (SEQ ID NOs: 122 and 123, and “AR”,respectively); hSG16.45 vH5 (SEQ ID NO: 31) with Kabat CDRs (SEQ ID NOs:152-154) and IMGT CDRs (SEQ ID NOs: 155-157).

FIG. 16 shows alignment of hSG16.45 heavy chain variants with human HVacceptor sequence HV3-9/HJ3. It shows the sequences of Rat SG16.45 vH(SEQ ID NO: 23) with Kabat CDRs (SEQ ID NOs: 110-112) and IMGT CDRs (SEQID NOs: 113-115); Hu HV3-9/HJ3 (SEQ ID NO: 26) with Kabat CDRs (SEQ IDNOs: 124 and 125) and IMGT CDRs (SEQ ID NOs: 126 and 127, and “AR”,respectively); hSG16.45 vH6 (SEQ ID NO: 32) with Kabat CDRs (SEQ ID NOs:158-160) and IMGT CDRs (SEQ ID NOs: 161-163).

FIG. 17 shows alignment of hSG16.45 heavy chain variants. It shows thesequences of hSG16.45 vH1-6 (SEQ ID NOs: 27-32).

FIG. 18 shows alignment of hSG16.45 light chain variants with human KVacceptor sequence KV3-20/KJ2. It shows the sequences of Rat SG16.45 vK(SEQ ID NO: 33) with Kabat CDRs (SEQ ID NOs: 164-166) and IMGT CDRs (SEQID NO: 167, “STS”, and SEQ ID NO: 168, respectively); Hu KV3-20/KJ2 (SEQID NO: 34) with Kabat CDRs (SEQ ID NOs: 169-171) and IMGT CDRs (SEQ IDNO: 172, “STS”, and SEQ ID NO: 173, respectively); hSG16.45 vK1 (SEQ IDNO: 35) with Kabat CDRs (SEQ ID NOs: 174-176) and IMGT CDRs (SEQ ID NO:177, “STS”, and SEQ ID NO: 178, respectively); hSG16.45 vK2 (SEQ ID NO:36) with Kabat CDRs (SEQ ID NOs: 179-181) and IMGT CDRs (SEQ ID NO: 182,“STS”, and SEQ ID NO: 183, respectively); hSG16.45 vK3 (SEQ ID NO: 37)with Kabat CDRs (SEQ ID NOs: 184-186) and IMGT CDRs (SEQ ID NO: 187,“STS”, and SEQ ID NO: 188, respectively); and hSG16.45 vK5 (SEQ ID NO:38) with Kabat CDRs (SEQ ID NOs: 189-191) and IMGT CDRs (SEQ ID NO: 192,“STS”, and SEQ ID NO: 193, respectively).

FIG. 19. shows alignment of hSG16.45 light chain variants. It shows thesequences of hSG16.45 vK1, vK2, vK3, vK5 (SEQ ID NOs: 35-38).

FIGS. 20A-C show in vivo activity of multi dosed hSG16.17-SEA in MM1Sdisseminated tumor model in SCID mice.

FIGS. 21A-C show in vivo activity of single dosed hSG16.17-SEA in EJMdisseminated tumor model in NSG mice.

FIG. 22 show in vivo activity of multi dosed hSG16.17-SEA inNCI-H929-luciferase disseminated tumor model in NSG mice.

FIGS. 23A-B show in vivo activity of single dosed hSG16.17-SEA inNCI-H929-luciferase disseminated tumor model in NSG mice.

FIG. 24 provides in vivo activity of single dosed hSG16.17-SEA inMOLP-8-luciferase disseminated tumor model in SCID mice.

FIG. 25 provides ADCC activity of the SG16.17 SEA antibody on MM1Rtarget cells.

DEFINITIONS

An “isolated” antibody refers to an antibody that has been identifiedand separated and/or recovered from components of its naturalenvironment and/or an antibody that is recombinantly produced. A“purified antibody” is an antibody that is typically at least 50% w/wpure of interfering proteins and other contaminants arising from itsproduction or purification but does not exclude the possibility that themonoclonal antibody is combined with an excess of pharmaceuticalacceptable carrier(s) or other vehicle intended to facilitate its use.Interfering proteins and other contaminants can include, for example,cellular components of the cells from which an antibody is isolated orrecombinantly produced. Sometimes monoclonal antibodies are at least60%, 70%, 80%, 90%, 95 or 99% w/w pure of interfering proteins andcontaminants from production or purification. The antibodies describedherein, including rat, chimeric, veneered and humanized antibodies canbe provided in isolated and/or purified form.

A “monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. (1991)Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597,for example or may be made by other methods. The antibodies describedherein are monoclonal antibodies.

Specific binding of a monoclonal antibody to its target antigen means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific bindingis detectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region, means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7, incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989), or acomposite of Kabat and Chothia, or IMGT, AbM or Contact or otherconventional definition of CDRs. Kabat also provides a widely usednumbering convention (Kabat numbering) in which corresponding residuesbetween different heavy chains or between different light chains areassigned the same number. Unless otherwise apparent from the context,Kabat numbering is used to designate the position of amino acids in thevariable regions. Unless otherwise apparent from the context EUnumbering is used to designated positions in constant regions.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, antibody fragments compete with the intact antibodyfrom which they were derived for specific binding to the targetincluding separate heavy chains, light chains Fab, Fab′, F(ab′)₂,F(ab)c, diabodies, Dabs, nanobodies, and Fv. Fragments can be producedby recombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes a diabody(homodimeric Fv fragment) or a minibody (V_(L)—V_(H)—C_(H)3), abispecific antibody or the like. A bispecific or bifunctional antibodyis an artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites (see, e.g., Songsivilai andLachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J.Immunol., 148:1547-53 (1992)).

The term “antibody” includes an antibody by itself (naked antibody) oran antibody conjugated to a cytotoxic or cytostatic drug.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions.

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% but preferably 75%,90% or 99% as measured in a competitive binding assay. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Antibodies that compete with the h2H12 antibody for binding to the humanBCMA protein are included in this disclosure.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Nonconservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

Designation of a range of values includes all integers within ordefining the range.

An antibody effector function refers to a function contributed by an Fcdomain(s) of an Ig. Such functions can be, for example,antibody-dependent cellular cytotoxicity, antibody-dependent cellularphagocytosis or complement-dependent cytotoxicity. Such function can beeffected by, for example, binding of an Fc effector domain(s) to an Fcreceptor on an immune cell with phagocytic or lytic activity or bybinding of an Fc effector domain(s) to components of the complementsystem. Typically, the effect(s) mediated by the Fc-binding cells orcomplement components result in inhibition and/or depletion of the BCMAtargeted cell. Fc regions of antibodies can recruit Fc receptor(FcR)-expressing cells and juxtapose them with antibody-coated targetcells. Cells expressing surface FcR for IgGs including FcγRIII (CD16),FcγRII (CD32) and FcγRIII (CD64) can act as effector cells for thedestruction of IgG-coated cells. Such effector cells include monocytes,macrophages, natural killer (NK) cells, neutrophils and eosinophils.Engagement of FcγR by IgG activates antibody-dependent cellularcytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).ADCC is mediated by CD16⁺ effector cells through the secretion ofmembrane pore-forming proteins and proteases, while phagocytosis ismediated by CD32⁺ and CD64⁺ effector cells (see Fundamental Immunology,4^(th) ed., Paul ed., Lippincott-Raven, N.Y., 1997, Chapters 3, 17 and30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al.,2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res.Treat. 53:199-207). In addition to ADCC and ADCP, Fc regions ofcell-bound antibodies can also activate the complement classical pathwayto elicit complement-dependent cytotoxicity (CDC). C1q of the complementsystem binds to the Fc regions of antibodies when they are complexedwith antigens. Binding of C1q to cell-bound antibodies can initiate acascade of events involving the proteolytic activation of C4 and C2 togenerate the C3 convertase. Cleavage of C3 to C3b by C3 convertaseenables the activation of terminal complement components including C5b,C6, C7, C8 and C9. Collectively, these proteins form membrane-attackcomplex pores on the antibody-coated cells. These pores disrupt the cellmembrane integrity, killing the target cell (see Immunobiology, 6^(th)ed., Janeway et al., Garland Science, N. Y., 2005, Chapter 2).

The term “antibody-dependent cellular cytotoxicity”, or ADCC, is amechanism for inducing cell death that depends upon the interaction ofantibody-coated target cells with immune cells possessing lytic activity(also referred to as effector cells). Such effector cells includenatural killer cells, monocytes/macrophages and neutrophils. Theeffector cells attach to an Fc effector domain(s) of Ig bound to targetcells via their antigen-combining sites. Death of the antibody-coatedtarget cell occurs as a result of effector cell activity.

The term “antibody-dependent cellular phagocytosis”, or ADCP, refers tothe process by which antibody-coated cells are internalized, either inwhole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an Fc effector domain(s)of Ig.

The term “complement-dependent cytotoxicity”, or CDC, refers to amechanism for inducing cell death in which an Fc effector domain(s) of atarget-bound antibody activates a series of enzymatic reactionsculminating in the formation of holes in the target cell membrane.Typically, antigen-antibody complexes such as those on antibody-coatedtarget cells bind and activate complement component C1q which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes.

A “cytotoxic effect” refers to the depletion, elimination and/or thekilling of a target cell. A “cytotoxic agent” refers to an agent thathas a cytotoxic effect on a cell.

Cytotoxic agents can be conjugated to an antibody or administered incombination with an antibody.

A “cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on acell, thereby inhibiting the growth and/or expansion of a specificsubset of cells. Cytostatic agents can be conjugated to an antibody oradministered in combination with an antibody.

The term “pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “pharmaceuticallycompatible ingredient” refers to a pharmaceutically acceptable diluent,adjuvant, excipient, or vehicle with which an anti-BCMA antibody isadministered to a subject.

The phrase “pharmaceutically acceptable salt,” refers topharmaceutically acceptable organic or inorganic salts of an anti-BCMA-1antibody or conjugate thereof or agent administered with an anti-BCMA-1antibody. Exemplary salts include sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p toluenesulfonate, and pamoate (i.e., 1,1′methylenebis-(2 hydroxy 3 naphthoate)) salts. A pharmaceutically acceptable saltmay involve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counterion. The counterion may be any organic orinorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counterion.

Unless otherwise apparent from the context, the term “about” encompassesinsubstantial variation having no significant effect on functionalproperties (e.g., within a margin of error or experimental measurement).

DETAILED DESCRIPTION I. General

The invention provides monoclonal antibodies that specifically bind toBCMA. The antibodies are useful for treatment and diagnoses of variouscancers and immunological disorders as well as detecting BCMA.

II. Target Molecules

Unless otherwise indicated, BCMA means a human BCMA. Exemplary humannucleic acid and amino acid sequences are provided by SEQ ID NOS:1 and2. Unless otherwise apparent from the context reference to BMCA means atleast an extracellular domain of the protein (approximately residues1-54 of SEQ ID NO: 2) and sometimes the complete protein. Likewise,unless otherwise apparent from the context reference to BAFF and APRILand their receptors other than BCMA refers to wild type human sequencese.g., as provided in the Swiss Prot Database.

III. Antibodies of the Invention

A. Binding Specificity and Functional Properties

The SG16.17 antibody is a rat monoclonal antibody that specificallybinds to human BCMA as described in the examples. An ATCC deposit wasmade on Aug. 15, 2005 under the Budapest Treaty. The ATCC is located at10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCCdeposit was assigned accession number of PTA-6937. The SG16.17 antibodyinhibits binding of BCMA to both of its ligands, APRIL and BAFF. TheSG16.17 antibody when linked to a human IgG1 elicits ADCC, binds to andelicits signaling through Fcγ receptors. The SG16.17 antibody can alsobe incorporated into an antibody drug conjugate to deliver a linked druginto the interior of cells expressing BCMA. The SG16.45 antibody isanother rat monoclonal antibody that specifically binds to human BCMA,inhibits its binding to its ligands and can deliver a linked drug to theinterior of cells expressing BCMA.

The invention provides humanized, chimeric and veneered forms of theSG16.17 antibody (designated hSG16.17, cSG16.17 or vSG16.17) and SG16.45(analogously designated). Such antibodies typically retain some or allof the properties for SG16.17 or SG16.45 noted above. For any givenproperty, humanized, chimeric or veneered antibodies may exhibit theproperty to the same extent within experimental error or more or lessthan rat SG16.17 or SG16.45. The affinity of humanized, chimeric orveneered forms of the rat SG16.17 antibody (i.e., Ka) can be greaterthan that of the rat SG16.17 antibody, or within a factor of five or afactor of two (i.e., more than or less than) than that of the ratSG16.17 antibody for human BCMA. Preferred humanized, chimeric orveneered SG16.17 antibodies bind to the same epitope and/or compete withrat SG16.17 antibodies for binding to human BCMA. The affinity ofhumanized, chimeric or veneered forms of the rat SG16.45 antibody (i.e.,Ka) can be greater than that of the rat SG16.45 antibody, or within afactor of five or a factor of two (i.e., more than or less than) thanthat of the rat SG16.45 antibody for human BCMA. Preferred humanized,chimeric or veneered SG16.45 antibodies bind to the same epitope and/orcompete with rat SG16.45 antibodies for binding to human BCMA.

Preferred humanized, chimeric and veneered antibodies inhibit cancer(e.g., growth of cells, metastasis and/or lethality to the organisms) orB-cell mediated immune disorders as shown in vitro, in an animal modelor clinical trial.

B. Antibodies

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. For humanization of SG16.17, a preferred acceptor sequence forthe heavy chain is the germline VH exon V_(H)1-2 and for the J exon(J_(H)), exon J_(H)-3. For the light chain, a preferred acceptorsequence is exon V_(L)1-12 and J exon J_(K)5. For humanization ofSG16.45, a preferred heavy chain acceptor sequence is HV3-23/HJ3 (SEQ IDNO: 24) and a preferred light chain acceptor sequence is KV3-20/KJ2 (SEQID NO: 34).

Thus, a humanized antibody is an antibody having at least four CDRsentirely or substantially from a non-human donor antibody and variableregion framework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Similarly a humanized heavychain has at least two and usually all three CDRs entirely orsubstantially from a donor antibody heavy chain, and a heavy chainvariable region framework sequence and heavy chain constant region, ifpresent, substantially from human heavy chain variable region frameworkand constant region sequences. Similarly a humanized light chain has atleast two and usually all three CDRs entirely or substantially from adonor antibody light chain, and a light chain variable region frameworksequence and light chain constant region, if present, substantially fromhuman light chain variable region framework and constant regionsequences. Other than nanobodies and dAbs, a humanized antibodycomprises a humanized heavy chain and a humanized light chain. A CDR ina humanized or human antibody is substantially from or substantiallyidentical to a corresponding CDR in a non-human antibody when at least60%, 85%, 90%, 95% or 100% of corresponding residues (as defined byKabat) are identical between the respective CDRs. The variable regionframework sequences of an antibody chain or the constant region of anantibody chain are substantially from a human variable region frameworksequence or human constant region respectively when at least 70%, 80%,85%, 90%, 95% or 100% of corresponding residues defined by Kabat areidentical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat, but alternatively as defined by IMGT, Chothia,composite Kabat-Chothia, AbM or Contact or other conventionaldefinition) from a mouse antibody, they can also be made with less thanall CDRs (e.g., at least 4, or 5) CDRs from a mouse antibody (e.g.,Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal ofMolecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol.36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441,2000).

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 Å of a CDR region); or    -   (4) mediates interaction between the heavy and light chains.

The invention provides humanized forms of the rat SG16.17 antibodyincluding six exemplified humanized heavy chain mature variable regions(hSG16.17 VH1-6) (SEQ ID Nos: 11-16) and four exemplified humanizedlight chain mature variable regions (hSG16.17 VK2-5) (SEQ ID NOs:19-22). The heavy and light chains can be combined in any permutations,with permutations including any of hSG16.17 VH1, VH3 or VH5 beingpreferred. The permutation having the best combination of bindingaffinity, percentage sequence identity to human germline, expression andpercentage of monomeric content was hSG16.17 VH3 VK2. This antibodyshows similar affinity within experimental error as the rat SG16.17,greater than 85% sequence identity with human germline in both heavy andlight chain variable regions (thus, qualifying for “humanized”designation under the new INN guideliness), high expression in CHOcells, and high proportion of monomers. Compared with most otherhumanized antibodies hSG16.17 VH3 VK2 is unusual in having a largenumber of variable region framework mutations in which human acceptorresidues are changed to the corresponding rat residue (13) but alsohaving a large number of “forward” CDR mutations, in which a rat residuein the Kabat CDRs is changed to the corresponding residue in the humanacceptor sequence, such that overall the antibody has sufficientsequence identity to human germline sequences to be classified ashumanized under INN guidelines. Most previous humanized antibodies havehad Kabat CDR entirely from the donor antibody.

The invention provides antibodies in which the heavy chain variableregion shows at least 90% identity to hSG16.17 VH3 (SEQ ID NO: 13) and alight chain variable region at least 90% identical to hSG16.17 VK2 (SEQID NO: 19). Some antibodies show at least 95%, 96%, 97%, 98% or 99%sequence identity to HV3 and at least 95%, 96%, 97%, 98% or 99% sequenceidentity to VK2. Some such antibodies include the the three Kabat CDRs(SEQ ID NOs: 60-62) of hSG16.17 VH3 (SEQ ID NO: 13) and three Kabat CDRs(SEQ ID NOs: 90-92) of hSG16.17 VK2 (SEQ ID NO: 19). Some suchantibodies include the the three Kabat CDRs (SEQ ID NOs: 60-62) ofhSG16.17 VH3 (SEQ ID NO: 13) and three Kabat CDRs (SEQ ID NOs: 90-92) ofhSG16.17 VK2 (SEQ ID NO: 19) provided that position H58 can be occupiedby N or K, position H60 can be occupied by A or N, position H61 can beoccupied by Q or E, position H62 can be occupied by K or N, position H64can be occupied by Q or K, position H65 can be occupied by G or T,position L24 can be occupied by R or L and position L53 can be occupiedby S or R. Preferably positions H58, H60, H61, H62, H64 and H65 areoccupied by N, A, Q, K, Q and G respectively and L24 and L53 areoccupied by R and S respectively. These recited residues represent aminoacids from a human acceptor sequence occupying positions within theKabat CDRs. Some antibodies have at least 1, 2, 3, 4, 5, 6, 7 or 8 ratresidues in the human Kabat CDRs replaced with corresponding residuesfrom a human acceptor sequence. In some antibodies positions H58, H60,H61, H62, H64 and H65 are occupied by N, A, Q, K, Q and G respectivelyand L24 and L53 are occupied by R and S respectively. Some antibodiesinclude at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14backmutations representing replacement of variable region human acceptorsequence residues with corrsponding rat residues.

In some antibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 ofpositions H20, H48, H69, H71, H73, H76, H80, H88, H91 and H93 areoccupied by L, I, M, A, K, N, V, A, F, and T respectively. In someantibodies at least 1, 2 or 3 of positions L46, L48 and L87 are occupiedby V, V and F respectively. In some antibodies, each of positions H20,H48, H69, H71, H73, H76, H80, H88, H91 and H93 are occupied by L, I, M,A, K, N, V, A, F, and T respectively and each of L46, L48 and L87 areoccupied by V, V and F respectively.

Insofar as humanized antibodies show any variation from the exemplifiedhSG16.17 VH3 VK2 humanized antibody, one possibility for such additionalvariation is additional backmutations in the variable region frameworks.Any or all of the positions backmutated in other exemplified humanizedheavy or light chain mature variable regions can also be made (i.e., 1,2, 3, 4, 5 or all 6) of H8 occupied by R, H67 occupied by A and H78occupied by A, L40 occupied by S, L78 occupied by M and L85 occupied byD, or all 5 of H38 occupied by N, H40 occupied by R, H73 occupied by K,H82A occupied by S, and H83 occupied by T in the heavy chain and 1 orboth of L3 occupied by K, and L20 occupied by I in the light chain.However, such additional backmutations are not preferred because they ingeneral do not improve affinity and introducing more mouse residues maygive increased risk of immunogenicity.

Another possible variation is to substitute more or fewer residues inthe CDRs of the mouse antibody with corresponding residues from humanCDRs sequences, typically from the CDRs of the human acceptor sequencesused in designing the exemplified humanized antibodies. In someantibodies only part of the CDRs, namely the subset of CDR residuesrequired for binding, termed the SDRs, are needed to retain binding in ahumanized antibody. CDR residues not contacting antigen and not in theSDRs can be identified based on regions of Kabat CDRs lying outside CDRsaccording to other definitions, such as Chothia hypervariable loops(Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/orempirically, or as described in Gonzales et al., Mol. Immunol. 41: 863(2004). In such humanized antibodies at positions in which one or moredonor CDR residues is absent or in which an entire donor CDR is omitted,the amino acid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

Although not preferred other amino acid substitutions can be made, forexample, in framework residues not in contact with the CDRs, or evensome potential CDR-contact residues amino acids within the CDRs. Oftenthe replacements made in the variant humanized sequences areconservative with respect to the replaced hSG16.17 VH3 VK2 amino.Preferably, replacements relative to hSG16.17 VH3 VK2 (whether or notconservative) have no substantial effect on the binding affinity orpotency of the humanized mAb, that is, its ability to bind human BCMAand inhibit growth of cancer cells.

Variants typically differ from the heavy and light chain mature variableregion sequences of hSG16.17 VH3 VK2 by a small number (e.g., typicallyno more than 1, 2, 3, 5 or 10 in either the light chain or heavy chainmature variable region, or both) of replacements, deletions orinsertions.

Other preferred combinations of humanized heavy and light chains includeany of hSG16.17 VH1 VK2, VH1 VK3, VH1 VK4, VH1 VK4, VH3 VK2, VH3 VK3,VH3 VK4, and VH3 VK5, and VH5 VK2, VH5 VK3, VH5 VK4, VH5 VK5, as well ashumanized antibodies in which the heavy and light chain variable regionsshow at least 90, 95, 96, 97, 98, or 99% identity with the heavy andlight chain variable regions of any of these antibodies.

The invention provides humanized forms of the rat SG16.45 antibodyincluding six exemplified humanized heavy chain mature variable regions(hSG16.45 VH1-6) (SEQ ID NOs: 27-32) and four exemplified humanizedlight chain mature variable regions (hSG16.45 VK1, 2, 3, and 5) (SEQ IDNOs: 35-38). The heavy and light chains can be combined in anypermutations, with permuations hSG16.45 VH5 VK2, VH1 VK1 and VH1 VK5being preferred. hSG16.45 HV5 VK2 shows greater than 85% sequenceidentity with human germline in both heavy and light chain variableregions (thus, qualifying for “humanized” designation under the new INNguideliness), high expression in CHO cells, a high proportion ofmonomers and adequate binding albeit slightly less than that of rat orchimeric SG16.45. hSG16.45 VH5 VK2 has 3 variable region backmutations(all in the heavy chain) and 3 Kabat CDR forward mutations, in which arat residue in the Kabat CDRs is changed to the corresponding residue inthe human acceptor sequence, such that overall the antibody hassufficient sequence identity to human germline sequences to beclassified as humanized under INN guidelines.

The invention provides antibodies in which the heavy chain variableregion shows at least 90% identity to hSG16.45 VH5 (SEQ ID NO: 31) and alight chain variable region at least 90% identical to hSG16.45 VK2. Someantibodies show at least 95%, 96%, 97%, 98% or 99% sequence identity tohSG16.45 VH5 and at least 95%, 96%, 97%, 98% or 99% sequence identity toVK2. Some such antibodies include the the three Kabat CDRs (SEQ ID NOs:152-154) of hSG16.45 VH5 (SEQ ID NO: 31) and three Kabat CDRs (SEQ IDNOs: 179-181) of hSG16.45 VK2 (SEQ ID NO: 36). Some such antibodiesinclude the the three Kabat CDRs (SEQ ID NOs: 152-154) of hSG16.45 VH5(SEQ ID NO: 31) and three Kabat CDRs (SEQ ID NOs: 179-181) of hSG16.45VK2 (SEQ ID NO: 36) provided that position H50 can be occupied by A or Sand position L24 can be occupied by R or L and position L26 can beoccupied by S or T. Preferably positions H50 is occupied by A andpositions L24 and L26 are occupied by R and S. These recited residuesrepresent amino acids from a human acceptor sequence occupying positionswithin the Kabat CDRs. Some antibodies have at least 1, 2, or 3 ratresidues in the human Kabat CDRs replaced with corresponding residuesfrom a human acceptor sequence. In some antibodies positions H50, L24and L26 are occupied by A, R and S respectively. Some antibodies includeat least 1, 2, or 3 backmutations representing replacement of variableregion human acceptor sequence residues with corrsponding rat residues.

In some antibodies at least 1, 2, or 3, of positions H30, H93 and H94are occupied by N, T and S respectively. In some antibodies, each ofpositions H30, H93 and H94 are occupied by N, T and S respectively

Insofar as humanized antibodies show any variation from the exemplifiedhSG16.45 VH5 VK2 humanized antibody, one possibility for such additionalvariation is additional backmutations in the variable region frameworks.Any or all of the positions backmutated in other exemplified humanizedheavy or light chain mature variable regions can also be made (i.e., 1,2, 3, or 4) of H37, H48, H76, H107 occupied by I, I, N, and Vrespectively and/or 1, 2, 3, 4, 5, 6 or 7 of L14, L19, L21, L38, L58,L71 and L78 occuiied by A, V, I, H, V, Y, and M respectively. However,such additional backmutations are not preferred because they in generaldo not improve affinity and introducing more mouse residues may giveincreased risk of immunogenicity.

Another possible variation is to substitute more or fewer residues inthe CDRs of the mouse antibody with corresponding residues from humanCDRs sequences, typically from the CDRs of the human acceptor sequencesused in designing the exemplified humanized antibodies. In someantibodies only part of the CDRs, namely the subset of CDR residuesrequired for binding, termed the SDRs, are needed to retain binding in ahumanized antibody. CDR residues not contacting antigen and not in theSDRs can be identified based on regions of Kabat CDRs lying outside CDRsaccording to other definitions, such as Chothia hypervariable loops(Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/orempirically, or as described in Gonzales et al., Mol. Immunol. 41: 863(2004). In such humanized antibodies at positions in which one or moredonor CDR residues is absent or in which an entire donor CDR is omitted,the amino acid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

Although not preferred other amino acid substitutions can be made, forexample, in framework residues not in contact with the CDRs, or evensome potential CDR-contact residues amino acids within the CDRs. Oftenthe replacements made in the variant humanized sequences areconservative with respect to the replaced hSG16.45 VH3 VK2. Preferably,replacements relative to hSG16.45 VH5 VK2 (whether or not conservative)have no substantial effect on the binding affinity or potency of thehumanized mAb, that is, its ability to bind human BCMA and inhibitgrowth of cancer cells.

Variants typically differ from the heavy and light chain mature variableregion sequences of SG16.45 VH5 VK2 by a small number (e.g., typicallyno more than 1, 2, 3, 5 or 10 in either the light chain or heavy chainmature variable region, or both) of replacements, deletions orinsertions.

Other preferred combinations of humanized heavy and light chains includeany of hSG16.45 VH1 VK1 and VH1 VK5, as well as humanized antibodies inwhich the heavy and light chain variable regions show at least 90, 95,96, 97, 98, or 99% identity with the heavy and light chain variableregions of any of these antibodies.

C. Selection of Constant Region

Heavy and light chain variable regions of humanized antibodies can belinked to at least a portion of a human constant region. The choice ofconstant region depends, in part, whether antibody-dependentcell-mediated cytotoxicity, antibody dependent cellular phagocytosisand/or complement dependent cytotoxicity are desired. For example, humanisotopes IgG1 and IgG3 have strong complement-dependent cytotoxicity,human isotype IgG2 weak complement-dependent cytotoxicity and human IgG4lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also inducestronger cell mediated effector functions than human IgG2 and IgG4.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv, oras single chain antibodies in which heavy and light chain variabledomains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype binds to a non-polymorphic region of a one or more otherisotypes. Exemplary wild type human kappa and IgG1 constant regionsequences (the latter with or without the C-terminal lysine) are providein SEQ ID NOS: 3-5.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004).

Exemplary substitution include the amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably an S239Cmutation in a human IgG1 isotype (numbering is according to the EU index(Kabat, Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987 and 1991); see US 20100158909,which is herein incorporated reference). Sequences of a heavy chainconstant regions with S239C with and without a C-terminal lysine areprovided by SEQ ID NOS: 6 and 7. The presence of an additional cysteineresidue allows interchain disulfide bond formation. Such interchaindisulfide bond formation can cause steric hindrance, thereby reducingthe affinity of the Fc region-FcγR binding interaction. The cysteineresidue(s) introduced in or in proximity to the Fc region of an IgGconstant region can also serve as sites for conjugation to therapeuticagents (i.e., coupling cytotoxic drugs using thiol specific reagentssuch as maleimide derivatives of drugs. The presence of a therapeuticagent causes steric hindrance, thereby further reducing the affinity ofthe Fc region-FcγR binding interaction. Other substitutions at any ofpositions 234, 235, 236 and/or 237 reduce affinity for Fcγ receptors,particularly FcγRI receptor (see, e.g., U.S. Pat. Nos. 6,624,821,5,624,821.) A preferred combination of mutations is S239D, A330L and1332E, which increases the affinity of the Fc domain for FcγRIIIA andconsequently increases ADCC.

The in vivo half-life of an antibody can also impact its effectorfunctions. The half-life of an antibody can be increased or decreased tomodify its therapeutic activities. FcRn is a receptor that isstructurally similar to MHC Class I antigen that non-covalentlyassociates with β2-microglobulin. FcRn regulates the catabolism of IgGsand their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellularvesicles) but not at pH 7.4 (pH of blood); this interaction enables IgGsto be recycled back to the circulation (Ghetie and Ward, 2000, Ann. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The region on human IgG1 involved in FcRn binding has been mapped(Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alaninesubstitutions at positions Pro238, Thr256, Thr307, GIn311, Asp312,Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields etal., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules harboring thesesubstitutions have longer serum half-lives. Consequently, these modifiedIgG1 molecules may be able to carry out their effector functions, andhence exert their therapeutic efficacies, over a longer period of timecompared to unmodified IgG1. Other exemplary substitutions forincreasing binding to FcRn include a Gln at position 250 and/or a Leu atposition 428. EU numbering is used for all positions in the constantregion.

Oligosaccharides covalently attached to the conserved Asn297 areinvolved in the ability of the Fc region of an IgG to bind FcγR (Lund etal., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1997, TrendsBiotechnol. 15:26-31). Engineering of this glycoform on IgG cansignificantly improve IgG-mediated ADCC. Addition of bisectingN-acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol.17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) to thisglycoform or removal of fucose (Shields et al., 2002, J. Biol. Chem.277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwaet al., 2004, Cancer Res. 64:2127-33) from this glycoform are twoexamples of IgG Fc engineering that improves the binding between IgG Fcand FcγR, thereby enhancing Ig-mediated ADCC activity.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fcregion has generated IgG variants with altered FcγR binding affinities(Shields et al., 2001, J. Biol. Chem. 276:6591-604). When compared toparental IgG1, a subset of these variants involving substitutions atThr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333/Lys334 toAla demonstrate increased in both binding affinity toward FcγR and ADCCactivity (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki etal., 2004, J. Mol. Biol. 336:1239-49).

Complement fixation activity of antibodies (both C1q binding and CDCactivity) can be improved by substitutions at Lys326 and Glu333(Idusogie et al., 2001, J. Immunol. 166:2571-2575). The samesubstitutions on a human IgG2 backbone can convert an antibody isotypethat binds poorly to C1q and is severely deficient in complementactivation activity to one that can both bind C1q and mediate CDC(Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methodshave also been applied to improve complement fixation activity ofantibodies. For example, the grafting of an 18-amino acidcarboxyl-terminal tail piece of IgM to the carboxyl-termini of IgGgreatly enhances their CDC activity. This is observed even with IgG4,which normally has no detectable CDC activity (Smith et al., 1995, J.Immunol. 154:2226-36). Also, substituting Ser444 located close to thecarboxy-terminal of IgG1 heavy chain with Cys induced tail-to-taildimerization of IgG1 with a 200-fold increase of CDC activity overmonomeric IgG1 (Shopes et al., 1992, J. Immunol. 148:2918-22). Inaddition, a bispecific diabody construct with specificity for C1q alsoconfers CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

Complement activity can be reduced by mutating at least one of the aminoacid residues 318, 320, and 322 of the heavy chain to a residue having adifferent side chain, such as Ala. Other alkyl-substituted non-ionicresidues, such as Gly, Ile, Leu, or Val, or such aromatic non-polarresidues as Phe, Tyr, Trp and Pro in place of any one of the threeresidues also reduce or abolish C1q binding. Ser, Thr, Cys, and Met canbe used at residues 320 and 322, but not 318, to reduce or abolish C1qbinding activity. Replacement of the 318 (Glu) residue by a polarresidue may modify but not abolish C1q binding activity. Replacingresidue 297 (Asn) with Ala results in removal of lytic activity but onlyslightly reduces (about three fold weaker) affinity for C1q. Thisalteration destroys the glycosylation site and the presence ofcarbohydrate that is required for complement activation. Any othersubstitution at this site also destroys the glycosylation site. Thefollowing mutations and any combination thereof also reduce C1q binding:D270A, K322A, P329A, and P311S (see WO 06/036291).

Reference to a human constant region includes a constant region with anynatural allotype or any permutation of residues occupying polymorphicpositions in natural allotypes. Also, up to 1, 2, 5, or 10 mutations maybe present relative to a natural human constant region, such as thoseindicated above to reduce Fcγ receptor binding or increase binding toFcRN.

D. Expression of Recombinant Antibodies

Humanized, chimeric or veneered antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines (e.g., DG44), variousCOS cell lines, HeLa cells, HEK293 cells, L cells, andnon-antibody-producing myelomas including Sp2/0 and NS0. Preferably, thecells are nonhuman. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

E. Glycosylation Variants

Antibodies may be glycosylated at conserved positions in their constantregions (Jefferis and Lund, (1997) Chem. Immunol. 65:111-128; Wright andMorrison, (1997) TibTECH 15:26-32). The oligosaccharide side chains ofthe immunoglobulins affect the protein's function (Boyd et al., (1996)Mol. Immunol. 32:1311-1318; Wittwe and Howard, (1990) Biochem.29:4175-4180), and the intramolecular interaction between portions ofthe glycoprotein which can affect the conformation and presentedthree-dimensional surface of the glycoprotein (Hefferis and Lund, supra;Wyss and Wagner, (1996) Current Opin. Biotech. 7:409-416).Oligosaccharides may also serve to target a given glycoprotein tocertain molecules based upon specific recognition structures. Forexample, it has been reported that in agalactosylated IgG, theoligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminalN-acetylglucosamine residues become available to bind mannose bindingprotein (Malhotra et al., (1995) Nature Med. 1:237-243). Removal byglycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinanthumanized murine monoclonal IgG1 antibody which recognizes the CDw52antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO)cells resulted in a complete reduction in complement mediated lysis(CMCL) (Boyd et al., (1996) Mol. Immunol. 32:1311-1318), while selectiveremoval of sialic acid residues using neuraminidase resulted in no lossof DMCL. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al. (1999) MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc.

Addition of glycosylation sites to the antibody can be accomplished byaltering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. These methods include isolation from a naturalsource (in the case of naturally-occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g., antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected. See, e.g., Hseet al., (1997) J. Biol. Chem. 272:9062-9070. In addition to the choiceof host cells, factors which affect glycosylation during recombinantproduction of antibodies include growth mode, media formulation, culturedensity, oxygenation, pH, purification schemes and the like. Variousmethods have been proposed to alter the glycosylation pattern achievedin a particular host organism including introducing or overexpressingcertain enzymes involved in oligosaccharide production (U.S. Pat. Nos.5,047,335; 5,510,261; 5,278,299). Glycosylation, or certain types ofglycosylation, can be enzymatically removed from the glycoprotein, forexample using endoglycosidase H (Endo H). In addition, the recombinanthost cell can be genetically engineered, e.g., make defective inprocessing certain types of polysaccharides. These and similartechniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides

A preferred form of modification of glycosylation of antibodies isreduced core fucosylation. “Core fucosylation” refers to addition offucose (“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at thereducing terminal of an N-linked glycan.

A “complex N-glycoside-linked sugar chain” is typically bound toasparagine 297 (according to the number of Kabat). As used herein, thecomplex N-glycoside-linked sugar chain has a biantennary composite sugarchain, mainly having the following structure:

where ± indicates the sugar molecule can be present or absent, and thenumbers indicate the position of linkages between the sugar molecules.In the above structure, the sugar chain terminal which binds toasparagine is called a reducing terminal (at right), and the oppositeside is called a non-reducing terminal. Fucose is usually bound toN-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by anα1,6 bond (the 6-position of GlcNAc is linked to the 1-position offucose). “Gal” refers to galactose, and “Man” refers to mannose.

A “complex N-glycoside-linked sugar chain” includes 1) a complex type,in which the non-reducing terminal side of the core structure has one ormore branches of galactose-N-acetylglucosamine (also referred to as“gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAcoptionally has a sialic acid, bisecting N-acetylglucosamine or the like;or 2) a hybrid type, in which the non-reducing terminal side of the corestructure has both branches of a high mannose N-glycoside-linked sugarchain and complex N-glycoside-linked sugar chain.

In some embodiments, the “complex N-glycoside-linked sugar chain”includes a complex type in which the non-reducing terminal side of thecore structure has zero, one or more branches ofgalactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and thenon-reducing terminal side of Gal-GlcNAc optionally further has astructure such as a sialic acid, bisecting N-acetylglucosamine or thelike.

According to the present methods, typically only a minor amount offucose is incorporated into the complex N-glycoside-linked sugarchain(s) of humanized, chimeric or veneered SG16.17 or SG16.45antibodies. For example, in various embodiments, less than about 60%,less than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,or less than about 3% of the molecules of an antibody have corefucosylation by fucose. In some embodiments, about 2% of the moleculesof the antibody has core fucosylation by fucose.

In certain embodiments, only a minor amount of a fucose analog (or ametabolite or product of the fucose analog) is incorporated into thecomplex N-glycoside-linked sugar chain(s). For example, in variousembodiments, less than about 60%, less than about 50%, less than about40%, less than about 30%, less than about 20%, less than about 15%, lessthan about 10%, less than about 5%, or less than about 3% of humanized,chimeric or veneered SG16.17 or SG16.45 antibodies have corefucosylation by a fucose analog or a metabolite or product of the fucoseanalog. In some embodiments, about 2% of humanized, chimeric or veneeredSG16.17 antibodies have core fucosylation by a fucose analog or ametabolite or product of the fucose analog.

Methods of making non-fucosylated antibodies by incubatingantibody-producing cells with a fucose analogue are described, e.g., inWO2009/135181. Briefly, cells that have been engineered to expresshumanized, chimeric or veneered SG16.17 antibodies antibody areincubated in the presence of a fucose analogue or an intracellularmetabolite or product of the fucose analog. An intracellular metabolitecan be, for example, a GDP-modified analog or a fully or partiallyde-esterified analog. A product can be, for example, a fully orpartially de-esterified analog. In some embodiments, a fucose analoguecan inhibit an enzyme(s) in the fucose salvage pathway. For example, afucose analog (or an intracellular metabolite or product of the fucoseanalog) can inhibit the activity of fucokinase, orGDP-fucose-pyrophosphorylase. In some embodiments, a fucose analog (oran intracellular metabolite or product of the fucose analog) inhibitsfucosyltransferase (preferably a 1,6-fucosyltransferase, e.g., the FUT8protein). In some embodiments, a fucose analog (or an intracellularmetabolite or product of the fucose analog) can inhibit the activity ofan enzyme in the de novo synthetic pathway for fucose. For example, afucose analog (or an intracellular metabolite or product of the fucoseanalog) can inhibit the activity of GDP-mannose 4,6-dehydratase or/orGDP-fucose synthetase. In some embodiments, the fucose analog (or anintracellular metabolite or product of the fucose analog) can inhibit afucose transporter (e.g., GDP-fucose transporter).

In one embodiment, the fucose analogue is 2-flurofucose. Methods ofusing fucose analogues in growth medium and other fucose analogues aredisclosed, e.g., in WO/2009/135181, which is herein incorporated byreference.

Other methods for engineering cell lines to reduce core fucosylationincluded gene knock-outs, gene knock-ins and RNA interference (RNAi). Ingene knock-outs, the gene encoding FUT8 (alpha 1,6-fucosyltransferaseenzyme) is inactivated. FUT8 catalyzes the transfer of a fucosyl residuefrom GDP-fucose to position 6 of Asn-linked (N-linked) GlcNac of anN-glycan. FUT8 is reported to be the only enzyme responsible for addingfucose to the N-linked biantennary carbohydrate at Asn297. Geneknock-ins add genes encoding enzymes such as GNTIII or a golgi alphamannosidase II. An increase in the levels of such enzymes in cellsdiverts monoclonal antibodies from the fucosylation pathway (leading todecreased core fucosylation), and having increased amount of bisectingN-acetylglucosamines. RNAi typically also targets FUT8 gene expression,leading to decreased mRNA transcript levels or knocking out geneexpression entirely. Any of these methods can be used to generate a cellline that would be able to produce a non-fucosylated antibody, e.g., ahumanized, chimeric or veneered SG16.17 antibody.

Many methods are available to determine the amount of fucosylation on anantibody. Methods include, e.g., LC-MS via PLRP-S chromatography andelectrospray ionization quadrupole TOF MS.

IV. Nucleic Acids

The invention further provides nucleic acids encoding any of thehumanized heavy and light chains described above. Typically, the nucleicacids also encode a signal peptide fused to the mature heavy and lightchains. Coding sequences on nucleic acids can be in operable linkagewith regulatory sequences to ensure expression of the coding sequences,such as a promoter, enhancer, ribosome binding site, transcriptiontermination signal and the like. The nucleic acids encoding heavy andlight chains can occur in isolated form or can be cloned into one ormore vectors. The nucleic acids can be synthesized by for example, solidstate synthesis or PCR of overlapping oligonucleotides. Nucleic acidsencoding heavy and light chains can be joined as one contiguous nucleicacid, e.g., within an expression vector, or can be separate, e.g., eachcloned into its own expression vector.

V. Antibody Drug Conjugates

Anti-MCMA antibodies can be conjugated to cytotoxic moieties to formantibody-drug conjugates (ADCs). Particularly suitable moieties forconjugation to antibodies are cytotoxic agents (e.g., chemotherapeuticagents), prodrug converting enzymes, radioactive isotopes or compounds,or toxins (these moieties being collectively referred to as therapeuticagents or drugs). For example, an anti-BCMA antibody can be conjugatedto a cytotoxic agent such as a chemotherapeutic agent, or a toxin (e.g.,a cytostatic or cytocidal agent such as, e.g., abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin). Examples of useful classesof cytotoxic agents include, for example, DNA minor groove binders, DNAalkylating agents, and tubulin inhibitors. Exemplary cytotoxic agentsinclude, for example, auristatins, camptothecins, duocarmycins,etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes,benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines (PBDs),indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vincaalkaloids. Techniques for conjugating therapeutic agents to proteins,and in particular to antibodies, are well-known. (See, e.g., Alley etal., Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J,2008, 14(3):154-169.)

The therapeutic agent (e.g., cytotoxic agent) can be conjugated to theantibody in a manner that reduces its activity unless it is detachedfrom the antibody (e.g., by hydrolysis, by antibody degradation, or by acleaving agent). Such therapeutic agent can be attached to the antibodyvia a linker. A therapeutic agent conjugated to a linker is alsoreferred to herein as a drug linker. The nature of the linker can varywidely. The components that make up the linker are chosen on the basisof their characteristics, which may be dictated in part, by theconditions at the site to which the conjugate is delivered.

The therapeutic agent can be attached to the antibody with a cleavablelinker that is sensitive to cleavage in the intracellular environment ofthe anti-BCMA-expressing cancer cell but is not substantially sensitiveto the extracellular environment, such that the conjugate is cleavedfrom the antibody when it is internalized by the anti-BCMA-expressingcancer cell (e.g., in the endosomal or, for example by virtue of pHsensitivity or protease sensitivity, in the lysosomal environment or inthe caveolear environment). The therapeutic agent can also be attachedto the antibody with a non-cleavable linker.

As indicated, the linker may comprise a cleavable unit. In some suchembodiments, the structure and/or sequence of the cleavable unit isselected such that it is cleaved by the action of enzymes present at thetarget site (e.g., the target cell). In other embodiments, cleavableunits that are cleavable by changes in pH (e.g. acid or base labile),temperature or upon irradiation (e.g. photolabile) may also be used.

In some embodiments, the cleavable unit may comprise one amino acid or acontiguous sequence of amino acids. The amino acid sequence may be thetarget substrate for an enzyme.

In some aspects, the cleavable unit is a peptidyl unit and is at leasttwo amino acids long. Cleaving agents can include cathepsins B and D andplasmin (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics83:67-123). Most typical are cleavable unit that are cleavable byenzymes that are present in anti-BCMA expressing cells, i.e., an enzymecleavable linker. Accordingly, the linker can be cleaved by anintracellular peptidase or protease enzyme, including a lysosomal orendosomal protease. For example, a linker that is cleavable by thethiol-dependent protease cathepsin-B, which is highly expressed incancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or aVal-Cit peptide or a Val-Ala peptide).

In some embodiments, the linker will comprise a cleavable unit (e.g., apeptidyl unit) and the cleavable unit will be directly conjugated to thetherapeutic agent. In other embodiments, the cleavable unit will beconjugated to the therapeutic agent via an additional functional unit,e.g., a self-immolative spacer unit or a non-self-immolative spacerunit. A non self-immolative spacer unit is one in which part or all ofthe spacer unit remains bound to the drug unit after cleavage of acleavable unit (e.g., amino acid) from the antibody drug conjugate. Toliberate the drug, an independent hydrolysis reaction takes place withinthe target cell to cleave the spacer unit from the drug.

With a self-immolative spacer unit, the drug is released without theneed for drug for a separate hydrolysis step. In one embodiment, whereinthe linker comprises a cleavable unit and a self immolative group, thecleavable unit is cleavable by the action of an enzyme and aftercleavage of the cleavable unit, the self-immolative group(s) release thetherapeutic agent. In some embodiments, the cleavable unit of the linkerwill be directly or indirectly conjugated to the therapeutic agent onone end and on the other end will be directly or indirectly conjugatedto the antibody. In some such embodiments, the cleavable unit will bedirectly or indirectly (e.g., via a self-immolative ornon-self-immolative spacer unit) conjugated to the therapeutic agent onone end and on the other end will be conjugated to the antibody via astretcher unit. A stretcher unit links the antibody to the rest of thedrug and/or drug linker. In one embodiment, the connection between theantibody and the rest of the drug or drug linker is via a maleimidegroup, e.g., via a maleimidocaproyl linker. In some embodiments, theantibody will be linked to the drug via a disulfide, for example thedisulfide linked maytansinoid conjugates SPDB-DM4 and SPP-DM1.

The connection between the antibody and the linker can be via a numberof different routes, e.g., through a thioether bond, through a disulfidebond, through an amide bond, or through an ester bond. In oneembodiment, the connection between the anti-BCMA antibody and the linkeris formed between a thiol group of a cysteine residue of the antibodyand a maleimide group of the linker. In some embodiments, the interchainbonds of the antibody are converted to free thiol groups prior toreaction with the functional group of the linker. In some embodiments, acysteine residue is an introduced into the heavy or light chain of anantibody and reacted with the linker. Positions for cysteine insertionby substitution in antibody heavy or light chains include thosedescribed in Published U.S. Application No. 2007-0092940 andInternational Patent Publication WO2008070593, each of which areincorporated by reference herein in its entirety and for all purposes.

In some embodiments, the antibody-drug conjugates have the followingformula I:

L−(LU−D)_(p)  (I)

wherein L is an anti-BCMA antibody, LU is a Linker unit and D is a Drugunit (i.e., the therapeutic agent). The subscript p ranges from 1 to 20.Such conjugates comprise an anti-BCMA antibody covalently linked to atleast one drug via a linker. The Linker Unit is connected at one end tothe antibody and at the other end to the drug.

The drug loading is represented by p, the number of drug molecules perantibody. Drug loading may range from 1 to 20 Drug units (D) perantibody. In some aspects, the subscript p will range from 1 to 20(i.e., both integer and non-integer values from 1 to 20). In someaspects, the subscript p will be an integer from 1 to 20, and willrepresent the number of drug-linkers on a singular antibody. In otheraspects, p represents the average number of drug-linker molecules perantibody, e.g., the average number of drug-linkers per antibody in areaction mixture or composition (e.g., pharmaceutical composition), andcan be an integer or non-integer value. Accordingly, in some aspects,for compositions (e.g., pharmaceutical compositions), p represents theaverage drug loading of the antibody-drug conjugates in the composition,and p ranges from 1 to 20.

In some embodiments, p is from about 1 to about 8 drugs per antibody. Insome embodiments, p is 1. In some embodiments, p is 2. In someembodiments, p is from about 2 to about 8 drugs per antibody. In someembodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4drugs per antibody. In some embodiments, p is about 2, about 4, about 6or about 8 drugs per antibody.

The average number of drugs per antibody unit in a preparation from aconjugation reaction may be characterized by conventional means such asmass spectroscopy, ELISA assay, HIC, and HPLC. The quantitativedistribution of conjugates in terms of p may also be determined.

Exemplary antibody-drug conjugates include auristatin basedantibody-drug conjugates, i.e., conjugates wherein the drug component isan auristatin drug. Auristatins bind tubulin, have been shown tointerfere with microtubule dynamics and nuclear and cellular division,and have anticancer activity. Typically the auristatin basedantibody-drug conjugate comprises a linker between the auristatin drugand the anti-BCMA antibody. The auristatins can be linked to theanti-BCMA antibody at any position suitable for conjugation to a linker.The linker can be, for example, a cleavable linker (e.g., a peptidyllinker) or a non-cleavable linker (e.g., linker released by degradationof the antibody). The auristatin can be auristatin E or a derivativethereof. The auristatin can be, for example, an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatins include MMAF (monomethylauristatin F), and MMAE (monomethyl auristatin E). The synthesis andstructure of exemplary auristatins are described in U.S. Pat. Nos.7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and U.S. Pat. No.7,968,687 each of which is incorporated herein by reference in itsentirety and for all purposes.

Exemplary auristatin based antibody-drug conjugates include vcMMAE,vcMMAF and mcMMAF antibody-drug conjugates as shown below wherein Ab isan antibody as described herein and val-cit represents thevaline-citrulline dipeptide:

or a pharmaceutically acceptable salt thereof. The drug loading isrepresented by p, the number of drug-linker molecules per antibody.Depending on the context, p can represent the average number ofdrug-linker molecules per antibody, also referred to the average drugloading. The variable p ranges from 1 to 20 and is preferably from 1 to8. In some preferred embodiments, when p represents the average drugloading, p ranges from about 2 to about 5. In some embodiments, p isabout 2, about 3, about 4, or about 5. In some aspects, the antibody isconjugated to the linker via a sulfur atom of a cysteine residue. Insome aspects, the cysteine residue is one that is engineered into theantibody. In other aspects, the cysteine residue is an interchaindisulfide cysteine residue.

Exemplary antibody-drug conjugates include PBD based antibody-drugconjugates; i.e., antibody-drug conjugates wherein the drug component isa PBD drug.

PBDs are of the general structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position, which is the electrophilic centerresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site. The ability of PBDs to form an adduct in the minorgroove enables them to interfere with DNA processing, hence their use asantitumor agents.

The biological activity of these molecules can be potentiated by joiningtwo PBD units together through their C8/C′-hydroxyl functionalities viaa flexible alkylene linker. The PBD dimers are thought to formsequence-selective DNA lesions such as the palindromic 5′-Pu-GATC-Py-3′interstrand cross-link, which is thought to be mainly responsible fortheir biological activity.

In some embodiments, PBD based antibody-drug conjugates comprise a PBDdimer linked to an anti-BCMA antibody. The monomers that form the PBDdimer can be the same or different, i.e., symmetrical or unsymmetrical.The PBD dimer can be linked to the anti-BCMA antibody at any positionsuitable for conjugation to a linker. For example, in some embodiments,the PBD dimer will have a substituent at the C2 position that providesan anchor for linking the compound to the anti-BCMA antibody. Inalternative embodiments, the N10 position of the PBD dimer will providethe anchor for linking the compound to the anti-BCMA antibody.

Typically the PBD based antibody-drug conjugate comprises a linkerbetween the PBD drug and the anti-BCMA antibody. The linker may comprisea cleavable unit (e.g., an amino acid or a contiguous sequence of aminoacids that is a target substrate for an enzyme) or a non-cleavablelinker (e.g., linker released by degradation of the antibody). Thelinker may further comprise a maleimide group for linkage to theantibody, e.g., maleimidocaproyl. The linker may, in some embodiments,further comprise a self-immolative group, such as, for example, ap-aminobenzyl alcohol (PAB) unit.

An exemplary PBD for use as a conjugate is described in InternationalApplication No. WO 2011/130613 and is as follows wherein the wavy lineindicates the site of attachment to the linker:

or a pharmaceutically acceptable salt thereof. An exemplary linker is asfollows wherein the wavy line indicates the site of attachment to thedrug and the antibody is linked via the maleimide group.

Exemplary PBDs based antibody-drug conjugates include antibody-drugconjugates as shown below wherein Ab is an antibody as described herein:

or a pharmaceutically acceptable salt thereof. The drug loading isrepresented by p, the number of drug-linker molecules per antibody.Depending on the context, p can represent the average number ofdrug-linker molecules per antibody, also referred to the average drugloading. The variable p ranges from 1 to 20 and is preferably from 1 to8. In some preferred embodiments, when p represents the average drugloading, p ranges from about 2 to about 5. In some embodiments, p isabout 2, about 3, about 4, or about 5. In some aspects, the antibody isconjugated to the drug linker via a sulfur atom of a cysteine residuethat is engineered into the antibody. In some aspects, the cysteineresidue is engineered into the antibody at position 239 (IgG1) asdetermined by the EU index (Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991).

VI. Animal Models of Immunological Disorders or BCMA-Expressing Cancers

The anti-BCMA antibodies or derivatives can be tested or validated inanimal models of immunological disorders or BCMA-expressing cancers.Examples for animal models of systemic and organ-specific autoimmunediseases including diabetes, lupus, systemic sclerosis, Sjögren'sSyndrome, experimental autoimmune encephalomyelitis (multiplesclerosis), thyroiditis, myasthenia gravis, arthritis, uveitis,inflammatory bowel disease have been described by Bigazzi, “AnimalModels of Autoimmunity: Spontaneous and Induced,” in The AutoimmuneDiseases (Rose and Mackay eds., Academic Press, 1998) and in “AnimalModels for Autoimmune and Inflammatory Disease,” in Current Protocols inImmunology (Coligan et al. eds., Wiley and Sons, 1997).

Allergic conditions, e.g., asthma and dermatitis, can also be modeled inrodents. Airway hypersensitivity can be induced in mice by ovalbumin(Tomkinson et al., 2001, J. Immunol. 166:5792-800) or Schistosomamansoni egg antigen (Tesciuba et al., 2001, J. Immunol. 167:1996-2003).The Nc/Nga strain of mice show marked increase in serum IgE andspontaneously develop atopic dermatitis-like leisons (Vestergaard etal., 2000, Mol. Med. Today 6:209-10; Watanabe et al., 1997, Int.Immunol. 9:461-66; Saskawa et al., 2001, Int. Arch. Allergy Immunol.126:239-47).

Injection of immuno-competent donor lymphocytes into a lethallyirradiated histo-incompatible host is a classical approach to induceGVHD in mice. Alternatively, the parent B6D2F1 murine model provides asystem to induce both acute and chronic GVHD. In this model the B6D2F1mice are F1 progeny from a cross between the parental strains of C57BL/6and DBA/2 mice. Transfer of DBA/2 lymphoid cells into non-irradiatedB6D2F1 mice causes chronic GVHD, whereas transfer of C57BL/6, C57131110or B10.D2 lymphoid cells causes acute GVHD (Slayback et al., 2000, BoneMarrow Transpl. 26:931-938; Kataoka et al., 2001, Immunology103:310-318).

Additionally, both human hematopoietic stem cells and mature peripheralblood lymphoid cells can be engrafted into SCID mice, and these humanlympho-hematopoietic cells remain functional in the SCID mice (McCune etal., 1988, Science 241:1632-1639; Kamel-Reid and Dick, 1988, Science242:1706-1709; Mosier et al., 1988, Nature 335:256-259). This hasprovided a small animal model system for the direct testing of potentialtherapeutic agents on human lymphoid cells. (See, e.g., Tournoy et al.,2001, J. Immunol. 166:6982-6991).

Moreover, small animal models to examine the in vivo efficacies of theanti-BCMA antibodies or derivatives can be created by implantingBCMA-expressing human tumor cell lines into appropriate immunodeficientrodent strains, e.g., athymic nude mice or SCID mice. Examples ofBCMA-expressing human lymphoma cell lines include, for example, Daudi(Ghetie et al., 1994, Blood 83:1329-36; Ghetie et al., 1990, Int. J.Cancer 15:481-85; de Mont et al., 2001, Cancer Res. 61:7654-59), Ramos(Ma et al., 2002, Leukemia 16:60-6; Press et al., 2001, Blood98:2535-43), HS-Sultan (Cattan and Maung, 1996, Cancer Chemother.Pharmacol. 38:548-52; Cattan and Douglas, 1994, Leuk. Res. 18:513-22),Raji (Ochakovskaya et al., 2001, Clin. Cancer Res. 7:1505-10; Breisto etal., 1999, Cancer Res. 59:2944-49), and CA46 (Kreitman et al., 1999,Int. J. Cancer 81:148-55). Non-limiting example of a BCMA-expressingHodgkin's lymphoma line is L540cy (Barth et al., 2000, Blood 95:3909-14;Wahl et al., 2002, Cancer Res. 62:3736-42). Non-limiting examples ofBCMA expressing human renal cell carcinoma cell lines include 786-O(Ananth et al., 1999, Cancer Res. 59:2210-16; Datta et al., 2001, CancerRes. 61:1768-75), ACHN (Hara et al., 2001, J. Urol. 166:2491-94; Miyakeet al., 2002, J. Urol. 167:2203-08), Caki-1 (Prewett et al., 1998, Clin.Cancer Res. 4:2957-66; Shi and Siemann, 2002, Br. J. Cancer 87:119-26),and Caki-2 (Zellweger et al., 2001, Neoplasia 3:360-67). Non-limitingexamples of BCMA-expressing nasopharyngeal carcinoma cell lines includeC15 and C17 (Burson et al., 1988, Int. J. Cancer 42:599-606; Bernheim etal., 1993, Cancer Genet. Cytogenet. 66:11-5). Non-limiting examples ofBCMA-expressing human glioma cell lines include U373 (Palma et al.,2000, Br. J. Cancer 82:480-7) and U87MG (Johns et al., 2002, Int. J.Cancer 98:398-408). These tumor cell lines can be established inimmunodeficient rodent hosts either as solid tumor by subcutaneousinjections or as disseminated tumors by intravenous injections. Onceestablished within a host, these tumor models can be applied to evaluatethe therapeutic efficacies of the anti-BCMA antibody or derivatives asdescribed herein on modulating in vivo tumor growth.

VII. Therapeutic Applications

The anti-BCMA antibodies of the invention can be used to treat cancer.Some such cancers show detectable levels of BCMA measured at either theprotein (e.g., by immunoassay using one of the exemplified antibodies)or mRNA level. Some such cancers show elevated levels of BCMA relativeto noncancerous tissue of the same type, preferably from the samepatient. An exemplary level of BCMA on cancer cells amenable totreatment is 5000-150000 BCMA molecules per cell, although higher orlower levels can be treated. Optionally, a level of BCMA in a cancer ismeasured before performing treatment.

Cancers treatable with antibodies of the invention include solid tumorsand hematological cancers, such as leukemias and lymphomas. Theantibodies are particularly suitable for cancers of B-cells. Examples ofcancers treatable with the antibodies include: adult and pediatric acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) andsecondary leukemia; non-Hodgkin's lymphoma (NHL) and Hodgkin's disease;myelodysplastic syndromes (MDS), myeloproliferative syndromes (MPS)multiple myeloma, Waldenstrom's macroglobulinemia or Burkett's lymphoma,malignant plasma cell neoplasms, BCMA+high-grade lymphoma, Kahler'sdisease and myelomatosis; plasma cell leukemia; plasmacytoma; B-cellprolymphocytic leukemia; hairy cell leukemia; follicular lymphoma(including follicular non-Hodgkin's lymphoma types); Burkitt's lymphoma(Endemic Burkitt's lymphoma; sporadic Burkitt's lymphoma): marginal zonelymphoma (Mucosa-Associated Lymphoid Tissue: MALT 1 MALToma; MonocytoidB cell lymphoma; splenic lymphoma with villous lymphocytes); mantle celllymphoma; large cell lymphoma (diffuse large cell; diffuse mixed cell;immunoblastic lymphoma; primary mediastinal B cell cymphoma;angiocentric lymphoma pulmonary B cell): small lymphocytic lymphoma(SLL); recursor B-lymphoblastic lymphoma; myeloid leukemia(granulocytic; myelogenous; acute myeloid leukemia; chronic myeloidleukemia; subacute myeloid leukemia; myeloid sarcoma; chloroma;granulocytic sarcoma; acute promyelocytic leukemia; acute myelomonocyticleukemia); Waldenstrom's macroglobulinemia, or other B-cell leukemia orlymphoma.

The antibodies of the invention are also useful for immune disordersmediated by immune cells expressing BCMA, particularly B-cell mediateddisorders. Examples of such diseases include rheumatoid arthritis,systemic lupus E (SLE), Type I diabetes, asthma, atopic dermitus,allergic rhinitis, thrombocytopenic purpura, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, and graft versus host disease immune-mediatedthrombocytopenia, haemolytic anaemia, bullous pemphigoid, myastheniagravis, Graves' disease, Addison's disease, pemphigus foliaceus,psoriasis, psoriatic arthritis, and ankylosing spondylitis.

Anti-BCMA antibodies alone or as drug-conjugates thereof, areadministered in an effective regime meaning a dosage, route ofadministration and frequency of administration that delays the onset,reduces the severity, inhibits further deterioration, and/or amelioratesat least one sign or symptom of cancer. If a patient is alreadysuffering from cancer, the regime can be referred to as atherapeutically effective regime. If the patient is at elevated risk ofthe cancer relative to the general population but is not yetexperiencing symptoms, the regime can be referred to as aprophylactically effective regime. In some instances, therapeutic orprophylactic efficacy can be observed in an individual patient relativeto historical controls or past experience in the same patient. In otherinstances, therapeutic or prophylactic efficacy can be demonstrated in apreclinical or clinical trial in a population of treated patientsrelative to a control population of untreated patients.

Exemplary dosages for a monoclonal antibody are 0.1 mg/kg to 50 mg/kg ofthe patient's body weight, more typically 1 mg/kg to 30 mg/kg, 1 mg/kgto 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12 mg/kg, or 1 mg/kg to 10mg/kg1, or 2 mg/kg to 30 mg/kg, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to 10 mg/kg, or 3 mg/kg to 30mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 12 mg/kg, or3 mg/kg to 10 mg/kg. Exemplary dosages for active monoclonal antibodydrug conjugates thereof, e.g., auristatins, are 1 mg/kg to 7.5 mg/kg, or2 mg/kg to 7.5 mg/kg or 3 mg/kg to 7.5 mg/kg of the subject's bodyweight, or 0.1-20, or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 mg/kg) or 10-1500 or 200-1500 mg as a fixed dosage.Exemplary dosages for highly active monoclonal antibody drug conjugatesthereof, e.g., PBDs, are 1.0 μg/kg to 1.0 mg/kg, or 1.0 μg/kg to 500.0μg/kg of the subject's body weight. In some methods, the patient isadministered then antibody or ADC every two, three or four weeks. Thedosage depends on the frequency of administration, condition of thepatient and response to prior treatment, if any, whether the treatmentis prophylactic or therapeutic and whether the disorder is acute orchronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Administration can also be localizeddirectly into a tumor. Administration into the systemic circulation byintravenous or subcutaneous administration is preferred. Intravenousadministration can be, for example, by infusion over a period such as30-90 min or by a single bolus injection.

The frequency of administration depends on the half-life of the antibodyor antibody-drug conjugate in the circulation, the condition of thepatient and the route of administration among other factors. Thefrequency can be daily, weekly, monthly, quarterly, or at irregularintervals in response to changes in the patient's condition orprogression of the cancer being treated. An exemplary frequency forintravenous administration is between twice a week and quarterly over acontinuous course of treatment, although more or less frequent dosing isalso possible. Other exemplary frequencies for intravenousadministration are between once weekly or once monthly over a continuouscourse of treatment, although more or less frequent dosing is alsopossible. For subcutaneous administration, an exemplary dosing frequencyis daily to monthly, although more or less frequent dosing is alsopossible.

The number of dosages administered depends on the nature of the canceror autoimmune disease (e.g., whether presenting acute or chronicsymptoms) and the response of the disorder to the treatment. For acutedisorders or acute exacerbations of a chronic disorder between 1 and 10doses are often sufficient. Sometimes a single bolus dose, optionally individed form, is sufficient for an acute disorder or acute exacerbationof a chronic disorder. Treatment can be repeated for recurrence of anacute disorder or acute exacerbation. For chronic disorders, an antibodycan be administered at regular intervals, e.g., weekly, fortnightly,monthly, quarterly, every six months for at least 1, 5 or 10 years, orthe life of the patient.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. The concentration of antibody in a liquid formulation can bee.g., 0.01-10 mg/ml, such as 1.0 mg/ml.

Treatment with antibodies of the invention can be combined withchemotherapy, radiation, stem cell treatment, surgery other treatmentseffective against the disorder being treated. Useful classes of otheragents that can be administered with antibodies to BCMA include, forexample, antibodies to other receptors expressed on cancerous cells,antitubulin agents (e.g., auristatins), DNA minor groove binders (e.g.,PBDs), DNA replication inhibitors, alkylating agents (e.g., platinumcomplexes such as cis-platin, mono(platinum), bis(platinum) andtri-nuclear platinum complexes and carboplatin), anthracyclines,antibiotics, antifolates, antimetabolites, chemotherapy sensitizers,duocarmycins, etoposides, fluorinated pyrimidines, ionophores,lexitropsins, nitrosoureas, platinols, pre-forming compounds, purineantimetabolites, puromycins, radiation sensitizers, steroids, taxanes,topoisomerase inhibitors, vinca alkaloids, and the like. The sameadditional treatments just mentioned for cancer can also be used forimmune mediated disorders. Additional agents for immune mediatedisorders include immune suppressors such as mast cell degranulationinhibitors, anti-histamines, corticosteroids, NSAIDs, azathioprine,cyclophosphamide, leukeran, and cyclosporine and biologicanti-inflammatory agents, such as Tysabri® or Humira®.

Treatment with anti-BCMA antibodies, optionally in combination with anyof the other agents or regimes described above alone or as an antibodydrug conjugate, can increase the median progression-free survival oroverall survival time of patients with cancer, especially when relapsedor refractory, by at least 30% or 40% but preferably 50%, 60% to 70% oreven 100% or longer, compared to the same treatment (e.g., chemotherapy)but without the anti-BCMA antibody. In addition or alternatively,treatment (e.g., standard chemotherapy) including the anti-BCMAantibody, alone or as an antibody-drug conjugate, can increase thecomplete response rate, partial response rate, or objective responserate (complete+partial) of patients with tumors by at least 30% or 40%but preferably 50%, 60% to 70% or even 100% compared to the sametreatment (e.g., chemotherapy) but without the anti-BCMA antibody.

Typically, in a clinical trial (e.g., a phase II, phase II/III or phaseIII trial), the aforementioned increases in median progression-freesurvival and/or response rate of the patients treated with standardtherapy plus the anti-BCMA antibody, relative to the control group ofpatients receiving standard therapy alone (or plus placebo), arestatistically significant, for example at the p=0.05 or 0.01 or even0.001 level. The complete and partial response rates are determined byobjective criteria commonly used in clinical trials for cancer, e.g., aslisted or accepted by the National Cancer Institute and/or Food and DrugAdministration.

VIII. Other Applications

The anti-BCMA antibodies disclosed herein can be used for detecting BCMAin the context of clinical diagnosis or treatment or in research.Expression of BCMA on a cancer provides an indication that the cancer isamenable to treatment with the antibodies of the present invention. Theantibodies can also be sold as research reagents for laboratory researchin detecting cells bearing BCMA and their response to various stimuli.In such uses, monoclonal antibodies can be labeled with fluorescentmolecules, spin-labeled molecules, enzymes or radioisotypes, and can beprovided in the form of kit with all the necessary reagents to performthe assay for BCMA. The antibodies can also be used to purify BCMAprotein, e.g., by affinity chromatography.

Any feature, step, element, embodiment, or aspect of the invention canbe used in combination with any other unless specifically indicatedotherwise. Although the present invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.

EXAMPLES Example 1: Antibody Development Preparation of Recombinant BCMAExtracellular Domain (BCMA ECD)

The extracellular domain (ECD) of human (amino acids 1-51) and mouseBCMA (amino acids 1-46) were cloned and expressed as a GST fusionprotein (pGEX4T1; Amersham Biosciences). Purified BCMA ECD was obtainedby capturing the BCMA fusion protein with glutathione-Sepharose andreleasing the BCMA ECD by protease digestion with thrombin. Thrombin wassubsequently removed by benzamidine sepharose.

Identification of BCMA Expression on Malignant B-Cell Lines

Quantitative flow cytometry was performed on multiple myeloma cell linesusing Vicky-1, a commercial antibody for BCMA (Alexis Biotechnology).Results showed that BCMA is prevalent among myeloma lines tested. NCIH929 showed positive cell surface staining for BCMA but lackedexpression of either BR3 or TACI. Because NCI H929 expressed BCMA butnot BR3 or TACI, it was used for cell-based screening of the BCMAhybridomas.

Development of a Transfected BCMA Cell Line.

Stable cell lines were developed by transfecting HEK 293 cells witheither a full-length BCMA clone or an empty vector. Flow cytometryconfirmed positive expression of BCMA on the surface of the BCMAtransfected (293: BCMA) but not the vector empty control plasmid (293:vector). These cell lines were subsequently used as a tool to confirmthe specificity of cloned BCMA antibodies.

Example 2: Immunization and Screening of Uncloned Hybridoma WellsImmunization and Screening of Antiserum

Our immunization strategy used amino acids 1-50 of the BCMA ECD so thatepitopes internal and external to ligand binding domain could betargeted by antibodies (FIGS. 1A and 1B) KLH-conjugated BCMA ECD wasgenerated from a commercial source (Alexis Biochemicals). Rats wereimmunized KLH-conjugated BCMA using Titermax adjuvant until a maximumimmune response was detected by ELISA. Immunized rats serum was alsoscreened for ability to block APRIL binding in a plate-based assay. Rat2-3 was selected for fusion because the antiserum had a significanttiter of human BCMA antibodies and it displayed robust blockingactivity.

Spleen cells from rat 2-3 were harvested, fused to X-63.Ag8.653.3.12.11mouse myeloma cells and selected as described (Goding, 1989). Culturesupernatants from the resulting hybridomas were screened by ELISA usingpurified hBCMA-GST (see flow chart in FIG. 2). Eighty positive wellswere identified and selected for expansion. Sixty of the eightypositives wells continued to showed an OD>0.5 by ELISA followingexpansion. These sixty uncloned hybridoma wells were then screened insecondary assays for cell-based binding, ligand blockade activity, andcross-reactivity to mouse BCMA. This led to the identification of twelvelead BCMA hybridoma wells. Cell binding data and ligand blockadeactivity from these twelve lead wells is summarized in FIG. 3. Hybridomawell 17 showed cell binding and ligand blockade activity that supersededthe commercial monoclonal Vicky-1 (Alexis Biochemicals). Eight wells(indicated with a red asterisk in FIG. 3) were taken forward for cloningbased on their ability to bind BCMA-positive cells or block ligandbinding.

Example 3: Characterization of Clonal Hybridomas Cell Binding and LigandBlockade Activity.

Hybridoma wells 11, 17, 20, 29, 40, 45 and 70 were taken through 2rounds of limited dilution cloning. From this point forward, theantibodies will be designated with the formal clone ID shown in Table 1.The specific binding of the antibodies to 293: BCMA cells but not to the293: vector control cells confirms that the antibodies are binding toBCMA.

TABLE 1 Formal Clone IDs. Uncloned Designation Cloned ID 11 SG16.11 17SG16.17 20 SG16.20 29 SG16.29 40 SG16.40 45 SG16.45 70 SG16.70

Ligand blockade activity of the new BCMA antibodies was compared usingsupernatant from the uncloned master well, supernatant from the clonedwell and purified antibody from a cloned well (FIG. 4). A commercialantibody was used as a positive control. SG-16.17 gave significantblocking of APRIL binding using culture supernatant from the clonedhybridoma well. A titration of the SG16.17 blockade of APRIL binding wasperformed in a separate experiment using purified SG16.17 and thecommercial antibody (FIG. 5). Purified SG16.17 displayed improvedblocking activity across similar concentrations when compared to thecommercial antibody. SG-16.45 showed dose-dependent inhibition of Aprilbinding although not as strongly as SG-16.17. Ligand blockade activityfor the remaining BCMA antibodies (SG-16.11, SG16.20, SG16.29, SG16.40,and SG16.70) was more modest. Certain blocking BCMA antibodies show >75%inhibition of APRIL binding as was observed with SG-16.17. More “modest”blocking antibodies including SG-16.11, SG-16.20, SG-16.29, SG-16.40,and SG-16.70 showed about 30% inhibition for APRIL binding (FIG. 4).

The ability of BAFF to bind immobilized BCMA was also analyzed in thepresence and absence of purified BCMA antibodies. Pretreatment with BCMAantibodies SG16.17, SG16.40, SG16.20 and SG17.70 all resulted in atitratable inhibition of BAFF binding to BCMA (FIG. 6). The relativeinhibition was determined by binding BAFF to immobilized BCMA in theabsence of antibody treatment (FIG. 6, asterisk). Taken together, thedata in FIGS. 5 and 6 shows that BCMA antibodies can block ligandbinding of APRIL and BAFF to BCMA and thereby interfere with B cellsurvival signals.

Example 4: Testing SG16.17 and SG16.45 Antibodies for ADCC andCytotoxicity as an ADC

The SG16.17 antibody was converted into a rat-human chimeric IgG byfusing the rat V_(H) and V_(L) domains to wild-type human IgG1 heavychain and K light chain constant domains, respectively. The chimerizedantibody, designated cSG16.17 wild-type, showed similar antigen bindingproperties when compared with the parental antibody SG16.17. Next, weinstalled Fc mutations, S239D:A330L:1332E, known to enhance ADCC, togenerate cSG16.17 mutant. Similar to cSG16.17 wild-type, generation ofthe Fc triple mutant did not alter the antigen-binding properties ofcSG16.17 mutant. Evaluation of cSG16.17 wild-type and cSG16.17 mutant inan ADCC assay with purified natural killer cells resulted indose-dependent lysis of JJN3 and U266 cells whereas no significant lysiswas observed with a nonbinding human IgG control. The cSG16.17 wild-typeantibody displayed limited ADCC activity on JJN3 cells, which wasincreased ˜100-fold in potency and >2-fold in efficacy (maximal lysis)by cSG16.17 mutant. Similarly, for U266 cells, the ADCC activity ofcSG16.17 mutant was enhanced ˜100-fold in potency and 2-fold in efficacycompared with the parent chimeric antibody. The concentration ofcSG16.17 mutant required for maximal lysis of both JJN3 and U266 cellswas ˜100 pmol/L. In contrast, the dissociation constant (K_(D)) ofcSG16.17 on JJN3 and U266 cells was estimated as 15 and 10 nmol/L,respectively. Thus, maximal lysis by cSG16.17 mutant was achieved atconcentrations well below those required to reach saturation binding.

We assessed the ability of SG16.17 and SG16.45 to induce cytotoxicity asADCs using vcMMAF with a stoichiometry of eight drugs per antibody.SG16.17 or SG16.45-vcMMAF8 was potently cytotoxic against H929 cells. Nodecline in cell viability was observed using a nonbinding control ADC orunconjugated antibodies. We also examined the potency of SG16.17 ADCacross other MM cell lines, including JJN3 and U266 cell lines.SG16.17-vcMMAF8 showed consistent and high potency (IC₅O values 130pmol/L) across all three MM cell lines whereas SG16.45-vcMMAF8 showedmore variability and less overall potency.

Example 5: Testing SG16.17 Antibody for Binding to FcγRIIIa, andSignaling Through FcγRIIIa

For the binding assay, CHO cells were transfected with FcγRIIIa (hCD16)and binding of labelled h00 antibody measured in competition withchimeric SG16.17 with wild type IgG1 and IgG1 S239D, A330L, I332Egenotype, and various IgG1 control antibodies. FIG. 12 shows thatchimeric SG16.17 competed more strongly than two control antibodies,rituximab and cOKT9. The mutant form of SG16.17 competed more stronglythan the wild type IgG1 form. The signaling assay uses U266 target cellsexpressing BCMA, Jurkat effector cells expressing FcγRIIIa andengineered to express a luciferase reporter from a NFAT response elementand Bio-Glo indicator. cSG16.17 G1 WT & S239D, A330L, 1332E bothelicited FcγRIIIa signaling with that from the S239D, A330L, I332E formbeing stronger (FIG. 13).

Example 6: Humanization of SG16.17

TABLE 2 Humanizing Mutations in hSG16.17 Heavy Chain Variants HV Exon vHAcceptor Donor Framework Acceptor Variant Sequence Residues CDR ResidueshvH1 HV1-2/HJ3 H8, H20, H48, H67, H69, H71, none H73, H76, H80, H88,H91, H93 hvH2 HV1-2/HJ3 H20, H48, H69, H71, H73, H34, H50, H58, H60,H76, H80, H88, H91, H93 H61, H62, H64, H65 hvH3 HV1-2/HJ3 H20, H48, H67,H69, H71, H58, H60, H61, H62, H73, H76, H80, H88, H91, H64, H65 H93 hvH4HV1-2/HJ3 H48, H67, H69, H71, H73, H34, H50, H58, H60, H76, H80, H88,H91, H93 H61, H62, H64, H65 hvH5 HV1-46/HJ3 H48, H67, H71, H73, H76,none H78, H80, H91, H93 hvH6 HV1-46/HJ3 H8, H20, H48, H71, H73, H76,none H78, H80, H91, H93

TABLE 3 Humanizing Mutations in hSG16.17 Kappa Light Chain Variants KVExon Donor Acceptor vK Variant Acceptor Sequence Framework Residues CDRResidues hVK2 KV1-12/KJ5 L46, L48, L87 L53 hVK3 KV1-12/KJ5 L46, L48, L87L24, L53 hVK4 KV1-12/KJ5 L46, L48, L78, L85, L87 none hVK5 KV1-12/KJ5L40, L46, L48, L87 L24, L53

TABLE 4 Specific Framework Mutations in hSG16.17 Heavy Chain VariantsVariant H8 H20 H48 H67 H69 H71 H73 H76 H78 H80 H88 H91 H93 % Human hvH1R* L* I* A* M* A* K* N* A V* A* F* T* 79.6 hvH2 G L* I* V M* A* K* N* AV* A* F* T* 88.8 hvH3 G L* I* A* M* A* K* N* A V* A* F* T* 86.7 hvH4 G VI* A* M* A* K* N* A V* A* F* T* 88.8 hvH5 G V I* A* M A* K* N* A* V* AF* T* 78.6 hvH6 R* L* I* V M A* K* N* A* V* A F* T* 85.7 *Rat residues

TABLE 5 Specific Framework Mutations in hSG16.17 Kappa Light ChainVariants Variant L40 L46 L48 L78 L85 L87 % Human hvK2 P V* V* L T F*86.3 hvK3 p V* V* L T F* 87.4 hvK4 p V* V* M* D* F* 83.2 hvK5 S* V* V* LT F* 86.3 *Rat residues

The rat heavy and light chain variable regions of the rat hybridomaexpressing SG16.17 were sequenced. HV1-2/HJ3 (SEQ ID NO: 9) orHV1-46/HJ3 (SEQ ID NO: 10) was used as the human acceptor sequence forthe heavy chain and KV1-12/KJ5 (SEQ ID NO: 18) was used as the humanacceptor sequence for the light chain.

Positions differing between rat donor and human acceptor sequencesincluded H8, H20, H48, H67, H69, H71, H76, H78, H80, H88, H91, H93, L40,L46, L48, L78, L85 and L87. Different permutations of these residueswere included as back mutations in different humanized heavy chain andlight chain sequences. Several rat residues in the Kabat CDRs were alsotested for replacement with corresponding residues of the human acceptorsequences. The positions of these residues were H34, H50, H58, H60, H61,H62, H64 and H65, and L24 and L53. Six humanized heavy chain variantsand four humanized light chain variants were designed and expressed.Tables 2 and 3 indicate the human acceptor sequence, back mutations(donor framework residues), and CDR substitutions (Acceptor CDRresidues) in each humanized variant chain. Tables 4 and 5 indicate theamino acids occupying each of the positions considered for back mutationin each of the humanized variant chain. These tables also indicate thepercent of residue identical to the closest human germline sequence.According to recent INN Guidelines only antibodies with at least 85%identity to a human germline sequence in both heavy and light chains canbe referred to as humanized. FIGS. 7-9 show alignments of humanizedheavy chain variable regions with the rat variable region and humanacceptor sequences. FIGS. 10 and 11 show alignment of the humanizedlight chain variable regions with the rat variable region and humanacceptor sequences. The C-terminal arginine (R) of the variable lightchains can alternatively be regarded as the N-terminal arginine of thelight chain constant region.

The six humanized heavy chains and four humanized light chains weretested in all 24 possible permutations for binding to BCMA expressed onNCI-H929 cells, which express about 50,000 molecules of BCMA per cell.The results are shown in Table 6 below. In brief, all of the humanizedlight chains showed good binding. Of the humanized heavy chains,variants VH1, VH3 and VH5 all showed improved binding compared witheither chimeric or rat SG16.17 antibody.

TABLE 6 Humanized Antibodies hSG16.17 Binding to BCMA Expressed onNCI-H929 Cells NCI-H929 hSG16.17 vH vK 3-pt Assay 1 vH1 vK2 ++++ 2 vH1vK3 ++++ 3 vH1 vK4 ++++ 4 vH1 vK5 ++++ 5 vH2 vK2 − 6 vH2 vK3 − 7 vH2 vK4− 8 vH2 vK5 − 9 vH3 vK2 ++++ 10 vH3 vK3 ++++ 11 vH3 vK4 ++++ 12 vH3 vK5++++ 13 vH4 vK2 − 14 vH4 vK3 − 15 vH4 vK4 − 16 vH4 vK5 − 17 vH5 vK2 ++++18 vH5 vK3 ++++ 19 vH5 vK4 ++++ 20 vH5 vK5 ++++ 21 vH6 vK2 ++ 22 vH6 vK3++ 23 vH6 vK4 ++ 24 vH6 vK5 ++ cSG16.17 +++ rSG16.17 +++

The humanized antibodies performing best on the NCI-H929 assay (i.e.,those containing VH1, VH3 or VH5 heavy chains, were further tested forbinding to U266 cells at a full range of concentration points. In thisassay, humanized antibodies containing VH1 heavy chains (regardless ofwhich humanized light chain variant was included) showed enhancedbinding relative to rat or chimeric SG16.17. Humanized antibodiescontaining VH3 or VH5 heavy chains (regardless of which humanized lightchain variant was included) showed the same binding within experimentalerror as rat or chimeric SG16.17 binding. Humanized antibodiescontaining VH2 or VH6 variable regions showed reduced binding relativeto rat or chimeric SG16.17 regardless of which humanized light chainvariant was included.

The humanized antibodies performing best on the NCI-H929 assay were alsocompared for protein expression level, monomer level and percentagesequence identity to human germ line as shown in Table 7 below.

TABLE 7 hBCMA Transient aSEC ≥85% human (vH, vK) Lead hSG16.17 vH vKBinding Titer (mg/L) (% Monomer) & INN Designation Selection 1 vH1 vK2++++ 139 90.4 79.6 86.3 Mix Y 2 vH1 vK3 ++++ 126 89.6 79.6 87.4 Mix Y 3vH1 vK4 ++++ 80 94.6 79.6 83.2 Chimeric N 4 vH1 vK5 ++++ 119 89.5 79.686.3 Mix N 9 vH3 vK2 ++++ 129 94.1 86.7 86.3 Humanized Y 10 vH3 vK3 ++++116 94.1 86.7 87.4 Humanized Y 11 vH3 vK4 ++++ 82 95.2 86.7 83.2 Mix Y12 vH3 vK5 ++++ 117 93.5 86.7 86.3 Humanized Y 17 vH5 vK2 ++++ 97 96.278.6 86.3 Mix Y 18 vH5 vK3 ++++ 86 96.1 78.6 87.4 Mix Y 19 vH5 vK4 ++++65 96.5 78.6 83.2 Chimeric N 20 vH5 vK5 ++++ 73 95.0 78.6 86.3 Mix Y

The VH3 VK2 humanized antibody was selected as the lead humanizedantibody based on it having the same binding affinity for human BCMA asrat and mouse SG16.17 antibodies (within experimental error); greaterthan 85% identity to human germline sequence in both heavy and lightchain variable regions, good expression and high percentage of monomers.

Example 7: Humanization of SG16.45

TABLE 8 Humanizing Mutations in hSG16.45 Heavy Chain Variants HV ExonAcceptor vH Acceptor CDR Variant Sequence Donor Framework ResiduesResidues hvH1 HV3-23/HJ3 H30, H37, H48, H93, H94, H107 none hvH2HV3-23/HJ3 H30, H37, H48, H93, H94, H107 H50, H60 hvH3 HV3-23/HJ3 H30,H37, H48, H76, H93, H94, H50, H60 H107 hvH4 HV3-23/HJ3 H30, H48, H76,H93, H94 H50 hvH5 HV3-74/HJ3 H30, H93, H94 H50 hvH6 HV3-9/HJ3 H30, H93,H94 H50, H60

TABLE 9 Humanizing Mutations in hSG16.45 Kappa Light Chain Variants KVExon vK Acceptor Donor Framework Acceptor Variant Sequence ResiduesResidues CDR hvK1 KV3-20/KJ2 L14, L19, L21. L38, L58, L71, L24, L26 L78hvK2 KV3-20/KJ2 none L24, L26 hvK3 KV3-20/KJ2 L21, L38, L58, L71 L24,L26 hvK5 KV3-20/KJ2 L38, L71 none

TABLE 10 Specific Framework Mutations in hSG16.45 Heavy Chain VariantsVariant H30 H37 H48 H76 H93 H94 H107 % Human hvH1 N* I* I* N T* S* V*86.5 hvH2 N* I* I* N T* S* V* 88.5 hvH3 N* I* I* S* T* S* V* 87.5 hvH4N* V I* S* T* S* T 87.5 hvH5 N* V V N T* S* T 88.5 hvH6 N* V V N T* S* T88.5 *Rat residues

TABLE 11 Specific Framework Mutations in hSG16.45 Kappa Light ChainVariants Variant L14 L19 L21 L38 L58 L71 L78 % Human hvK1 A* V* I* H* V*Y* M* 79.2 hvK2 L A L Q I F L 86.5 hvK3 L A I* H* V* Y* L 82.3 hvK5 L AL H* I Y* L 82.3 *Rat residues

The rat heavy and light chain variable regions of the rat hybridomaexpressing SG16.45 were sequenced. HV3-23/HJ3 (SEQ ID NO: 24) was usedas the human acceptor sequence for the heavy chain and KV3-20/KJ2 (SEQID NO: 34) was used as the human acceptor sequence for the light chain.

Variable region framework positions differing between rat donor andhuman acceptor sequences included H30, H37, H48, H67, H93, H94 and H107and positions L14, L19, L21, L38, L58, L71 and L78. Differentpermutations of these residues were included as back mutations indifferent humanized heavy chain and light chain sequences. Several ratresidues in the Kabat CDRs were also tested for replacement withcorresponding residues of the human acceptor sequences. The positions ofthese residues were H50, H60, L24 and L26. Six humanized heavy chainvariants and four humanized light chain variants were designed andexpressed. Tables 8 and 9 indicate the human acceptor sequence, backmutations (donor framework residues), and CDR substitutions (AcceptorCDR residues) in each humanized variant chain. Tables 10 and 11 indicatethe amino acids occupying each of the positions considered for backmutation in each of the humanized variant chain. These tables alsoindicate the percent of residue identical to the closest human germlinesequence. According to recent INN Guidelines only antibodies with atleast 85% identity to a human germline sequence in both heavy and lightchains can be referred to as humanized. FIGS. 14-17 show an alignment ofhumanized heavy chain variable regions with the rat variable region andhuman acceptor sequences. FIGS. 18 and 19 show alignments of the lightchain variable regions. The C-terminal arginine (R) of the variablelight chains can alternatively be regarded as the N-terminal arginine ofthe light chain constant region.

The six humanized heavy chains and four humanized light chains weretested in all 24 possible permutations for binding to BCMA expressed onNCI-H929 cells, which express about 50,000 molecules of BCMA per cell.The results are shown in Table 12 below.

TABLE 12 Humanized Antibodies hSG16.45 Binding to BCMA Expressed onNCI-H929 Cells NCI-H929 hSG16.45 vH vK 3-pt Assay 1 vH1 vK1 +++ 2 vH1vK2 +++ 3 vH1 vK3 +++ 4 vH1 vK5 +++ 5 vH2 vK1 − 6 vH2 vK2 − 7 vH2 vK3 −8 vH2 vK5 − 9 vH3 vK1 − 10 vH3 vK2 − 11 vH3 vK3 − 12 vH3 vK5 ++ 13 vH4vK1 + 14 vH4 vK2 + 15 vH4 vK3 + 16 vH4 vK5 ++ 17 vH5 vK1 ++ 18 vH5 vK2++ 19 vH5 vK3 ++ 20 vH5 vK5 ++ 21 vH6 vK1 + 22 vH6 vK2 + 23 vH6 vK3 + 24vH6 vK5 ++ cSG16.45 +++ rSG16.45 +++

The humanized antibodies performing best on the NCI-H929 assay, werefurther tested for binding to U266 cells at a full range ofconcentration points, as well as for expression and monomer content, aswell as sequence identity to human germline (Table 13).

TABLE 13 hSG16.45 VH VK hBCMA IgG mg aSEC % VH % VK % INN 1 VH1 VK1 +++0.67 94.5 86.5 79.2 Mix 3 VH1 VK3 +++ 0.54 94.6 86.5 82.3 Mix 4 VH1 VK5+++ 0.16 76.0 86.5 82.3 Mix 17 VH5 VK1 ++ 0.64 94.4 88.5 79.2 Mix 18 VH5VK2 ++ 0.65 93.7 88.5 86.5 Hu 19 VH5 VK3 ++ 0.64 94.1 88.5 82.3 Mix

The VH5 VK2, VH1 VK1 and VH1 VK3 were the best antibodies overall basedon binding affinity for human, sequence identity to human germlinesequence in both heavy and light chain variable regions, good expressionand high percentage of monomers VH1 VK1 and VH1 VK3 have somewhat higherbinding (the same as rat or chimeric within experimental error) butlower sequence identity to human germline.

Example 8: Synthesis of a Reduced-Fucosylated hSG16.17 or hSG16.45Antibody

The hSG16.17 VH3 VK2 or hSG16.45 VH5 VK2 antibody was expressed in CHOcells. A fucosylation inhibitor, 2-fluorofucose, was included in thecell culture media during the production of antibodies resulted innon-fucosylated antibody. See, e.g., Okeley et al., Proc. WWI Acad. Sci.110:5404-55409 (2013). The base media for cell growth was fucose freeand 2-flurofucose was added to the media to inhibit proteinfucosylation. Ibid. Incorporation of fucose into antibodies was measuredby LC-MS via PLRP-S chromatography and electrospray ionization quadropleTOF MS. Ibid.

Example 9: In Vivo Activity of hSG16.17-SEA in SCID or NSG Mice

FIGS. 20A-C showed in vivo activity of multi dosed hSG16.17-SEA in MM1Sdisseminated tumor model in SCID mice. Animals were implanted with MM1Scells IV, and antibody dosing was initiated 9 days post implant. Animalsurvival was followed over time. N=8 animals per group. BCMA copy#=7,000, CD38 copy #=14,000. A) 1 mg/kg weekly ip for 5 weeks B) 3 mg/kgweekly ip for 5 weeks and C) 10 mg/kg weekly ip for 5 weeks. SCIDanimals contain effector cells to mediate ADCC and ADCP. Data in thisfigure show that hSG16.17 SEA improves survival comparable todaratumumab (CD38 targeted Ab. Non-binding h00 control showed noactivity.

FIGS. 21A-C showed In vivo activity of single dosed hSG16.17-SEA in EJMdisseminated tumor model in NSG mice. NSG animals contain no NK cellsand minimally active macrophages Animals were implanted with EJM cellsIV, and a single dose of antibody was given ip 5 days post implant.Animal survival was followed over time. N=8 animals per group. BCMA copy#=45,000. CD38 copy #=47,000. CS1 copy #=14,000. A) 1 mg/kg dose B) 3mg/kg dose C) 10 mg/kg dose. Data in this figure show that hSG16.17 SEAincreases survival to an equal or greater extent than daratumumab (CD38targeted Ab) and elotuzumab (CS1 targeted Ab). WT SG16.17 can alsoinduce increased survival. Non-binding h00 control showed no activity atthe highest dose. Since there are minimal effector cells in theseanimals, activity of WT and SEA hSG16.17 antibodies is likely due toblocking of the APRIL and BAFF proliferation signals.

FIG. 22 showed in vivo activity of multi dosed hSG16.17-SEA inNCI-H929-luciferase disseminated tumor model in NSG mice. NSG animalswere implanted with NCI-H929 luciferase cells. Antibody dosing wasinitiated 21 days post implant when bioluminescence was observed in thebone marrow. Dosed ip weekly for 5 doses total. N=5 animals per group.BCMA copy #=25,000. CD38 copy #=45,000. CS1 copy #=3,000. Averageluminescence is plotted over time in comparison to untreated and naïveanimals. hSG16.17 SEA displayed much better activity compared todaratumumab (CD38 targeted Ab) and elotuzumab (CS1 targeted Ab). Theincreased luminescence observed in the hSG16.17-SEA 10 mg/kg group isdriven by a single animal.

FIGS. 23A and 23B showed in vivo activity of single dosed hSG16.17-SEAin NCI-H929-luciferase disseminated tumor model in NSG mice. NSG animalswere implanted with NCI-H929 luciferase cells. Antibody dosing wasinitiated 21 days post injection when bioluminescence was observed inthe bone marrow. Dosed once IP. N=5 animals per group. A) 3 mg/kg WT vsSEA antibodies. B) Dose range of hSG16.17 SEA. Data in this figure showthat hSG16.17 SEA can be active at 0.3 mg/kg single dose and hSG16.17SEAcan be more active than its WT (fucosylated) counterpart.

FIGS. 23A and 23B showed in vivo activity of single dosed hSG16.17-SEAin NCI-H929-luciferase disseminated tumor model in NSG mice. NSG animalswere implanted with NCI-H929 luciferase cells. Antibody dosing wasinitiated 21 days post injection when bioluminescence was observed inthe bone marrow. Dosed once IP. N=5 animals per group. A) 3 mg/kg WT vsSEA antibodies. B) Dose range of hSG16.17 SEA. Data in this figure showthat hSG16.17 SEA can be active at 0.3 mg/kg single dose and hSG16.17SEAcan be more active than its WT (fucosylated) counterpart. Effects onluminescence translates to prolonged animal survival (data not shown).

FIG. 24 In vivo activity of single dosed hSG16.17-SEA inMOLP-8-luciferase disseminated tumor model in SCID mice. SCID animalswere implanted with MOLP-8 luciferase cells by IV. Antibody dosing wasinitiated 13 days post injection when bioluminescence was observed inthe bone marrow. Dosed once IP. N=5 animals per group. BCMA copy#=2,000. Luminescence is plotted over time. These data show that evenwith only 2000 BCMA copies the hSG16.17-SEA displays significantantitumor activity. Deglycosylated SEA BCMA antibody, which does notbind FcγRII or FcγRIII, showed no activity similar to h00 SEAnon-binding control. This reveals the importance of Fc mediated activityin this model.

FIG. 25 The SG16.17 SEA antibody displays improved ADCC activity on MM1Rtarget cells in comparison to WT antibody in vitro. NK cells wereisolated from PBMCs via negative selection using an EasySep Human NKcell enrichment kit, and resulting CD16+ cells were quantitated.Multiple myeloma MM1R ADCC target cells were labeled with chromium-51for 1 hr. A dilution series of antibodies was added to the assay plate,followed by target cells (T) and NK effector cells (E) at a 13:1 E:Tratio. Lysis was calculated based on total and spontaneous releasecontrols after 4 hrs at 37° C. These data show a significant improvementin ADCC activity of the afucosylated SEA SG16.17 antibody over WTantibody as well as clinical antibodies, daratumumba and elotuzumab.

Although the invention has been described in detail for purposes ofclarity of understanding, certain modifications may be practiced withinthe scope of the appended claims. All publications including accessionnumbers, websites and the like, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent difference version of a sequence, website or otherreference may be present at different times, the version associated withthe reference at the effective filing date is meant. The effectivefiling date means the earliest priority date at which the accessionnumber at issue is disclosed. Unless otherwise apparent from the contextany element, embodiment, step, feature or aspect of the invention can beperformed in combination with any other.

1-6. (canceled)
 7. A method for treating a subject with a cancer thatexpresses human B-cell maturation antigen (BCMA), the method comprisingadministering to the subject an effective amount of an antibody or abinding fragment thereof that binds to human BCMA, wherein the antibodyor binding fragment comprises a mature heavy chain variable region and amature light chain variable region, wherein the mature heavy chainvariable region comprises complementarity determining regions (CDRs)comprising the amino acid sequences of SEQ ID NOs:60, 61 and 62, and themature light chain variable region comprises CDRs comprising the aminoacid sequences of SEQ ID NOs:90, 91 and
 92. 8. The method of claim 7,wherein the mature heavy chain variable region is fused to a heavy chainconstant region and the mature light chain variable region is fused to alight chain constant region.
 9. The method of claim 8, wherein the heavychain constant region is a mutant form of a natural human constantregion and has reduced binding to an Fcγ receptor relative to thenatural human constant region.
 10. The method of claim 8, wherein theheavy chain constant region is of immunoglobulin G1 (IgG1) isotype. 11.The method of claim 8, wherein the heavy chain constant region comprisesthe amino acid sequence of SEQ ID NO:5 and the light chain constantregion comprises the amino acid sequence of SEQ ID NO:3.
 12. The methodof claim 7, wherein the antibody or antibody binding fragment isnon-fucosylated.
 13. The method of claim 7, wherein the antibody orantibody binding fragment is an antibody binding fragment.
 14. Themethod of claim 13, wherein the antibody binding fragment is selectedfrom the group consisting of a Fab, a Fab′, and a F(ab′)₂.
 15. Themethod of claim 7, wherein the antibody is a humanized antibody.
 16. Themethod of claim 7, wherein the cancer is a hematological cancer.
 17. Themethod of claim 16, wherein the hematological cancer is a myeloma,leukemia or a lymphoma.
 18. The method of claim 16, wherein thehematological cancer is multiple myeloma.
 19. The method of claim 16,wherein the hematological cancer is non-Hodgkin's lymphoma (NHL) orHodgkin's lymphoma.
 20. The method of claim 16, wherein thehematological cancer is myelodysplastic syndromes (MDS),myeloproliferative syndromes (MPS), Waldenström's macroglobulinemia orBurkett's lymphoma.
 21. A method for treating a subject with a cancerthat expresses human B-cell maturation antigen (BCMA), the methodcomprising administering to the subject an effective amount of anantibody or a binding fragment thereof that binds to human BCMA, whereinthe antibody or binding fragment comprises a mature heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:13, and a maturelight chain variable region comprising the amino acid sequence of SEQ IDNO:19.
 22. The method of claim 21, wherein the mature heavy chainvariable region is fused to a heavy chain constant region and the maturelight chain variable region is fused to a light chain constant region.23. The method of claim 22, wherein the heavy chain constant region is amutant form of a natural human constant region and has reduced bindingto an Fcγ receptor relative to the natural human constant region. 24.The method of claim 22, wherein the heavy chain constant region is ofimmunoglobulin G1 (IgG1) isotype.
 25. The method of claim 22, whereinthe heavy chain constant region comprises the amino acid sequence of SEQID NO:5 and the light chain constant region comprises the amino acidsequence of SEQ ID NO:3.
 26. The method of claim 21, wherein theantibody or antibody binding fragment is non-fucosylated.
 27. The methodof claim 21, wherein the antibody or antibody binding fragment is anantibody binding fragment.
 28. The method of claim 21, wherein thecancer is a hematological cancer.
 29. The method of claim 28, whereinthe hematological cancer is a myeloma, leukemia or a lymphoma.
 30. Themethod of claim 28, wherein the hematological cancer is multiplemyeloma.
 31. The method of claim 28, wherein the hematological cancer isnon-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma.
 32. A method fortreating a subject with a cancer that expresses human B-cell maturationantigen (BCMA), the method comprising administering to the subject aneffective amount of an antibody that binds to human BCMA, wherein (a)the antibody is a monoclonal, immunoglobulin G1 (IgG1) antibody; and (b)the antibody comprises a mature heavy chain variable region and a maturelight chain variable region, wherein the mature heavy chain variableregion comprises complementarity determining regions (CDRs) comprisingthe amino acid sequences of SEQ ID NOs:60, 61 and 62, and the maturelight chain variable region comprises CDRs comprising the amino acidsequences of SEQ ID NOs:90, 91 and
 92. 33. The method of claim 32,wherein the cancer is a hematological cancer.
 34. The method of claim33, wherein the hematological cancer is a myeloma, leukemia or alymphoma.
 35. The method of claim 33, wherein the hematological canceris multiple myeloma.
 36. The method of claim 33, wherein thehematological cancer is non-Hodgkin's lymphoma (NHL) or Hodgkin'slymphoma.
 37. A method for treating a subject with a cancer thatexpresses human B-cell maturation antigen (BCMA), the method comprisingadministering to the subject an effective amount of an antibody thatbinds to human BCMA, wherein (a) the antibody is a monoclonal,immunoglobulin G1 (IgG1) antibody; and (b) the antibody comprises amature heavy chain variable region comprising the amino acid sequence ofSEQ ID NO:13, and a mature light chain variable region comprising theamino acid sequence of SEQ ID NO:19.
 38. The method of claim 37, whereinthe cancer is a hematological cancer.
 39. The method of claim 38,wherein the hematological cancer is a myeloma, leukemia or a lymphoma.40. The method of claim 38, wherein the hematological cancer is multiplemyeloma.
 41. The method of claim 38, wherein the hematological cancer isnon-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma.
 42. A method fortreating a subject with a cancer that expresses human B-cell maturationantigen (BCMA), the method comprising administering to the subject aneffective amount of a monoclonal, non-fucosylated antibody that binds tohuman BCMA, wherein the antibody comprises a mature heavy chain variableregion and a mature light chain variable region, wherein the matureheavy chain variable region comprises complementarity determiningregions (CDRs) comprising the amino acid sequences of SEQ ID NOs:60, 61and 62, and the mature light chain variable region comprises CDRscomprising the amino acid sequences of SEQ ID NOs:90, 91 and
 92. 43. Themethod of claim 42, wherein the antibody is an immunoglobulin G1 (IgG1)antibody.
 44. The method of claim 42, wherein the mature heavy chainvariable region is fused to a heavy chain constant region and the maturelight chain variable region is fused to a light chain constant region,and wherein the heavy chain constant region comprises the amino acidsequence of SEQ ID NO:5 and the light chain constant region comprisesthe amino acid sequence of SEQ ID NO:3.
 45. The method of claim 42,wherein the cancer is a hematological cancer.
 46. The method of claim45, wherein the hematological cancer is a myeloma, leukemia or alymphoma.
 47. The method of claim 45, wherein the hematological canceris multiple myeloma.
 48. The method of claim 45, wherein thehematological cancer is non-Hodgkin's lymphoma (NHL) or Hodgkin'slymphoma.
 49. A method for treating a subject with a cancer thatexpresses human B-cell maturation antigen (BCMA), the method comprisingadministering to the subject an effective amount of a monoclonal,non-fucosylated antibody that binds to human BCMA, wherein the antibodycomprises a mature heavy chain variable region comprising the amino acidsequence of SEQ ID NO:13, and a mature light chain variable regioncomprising the amino acid sequence of SEQ ID NO:19.
 50. The method ofclaim 49, wherein the cancer is a hematological cancer.
 51. The methodof claim 50, wherein the hematological cancer is a myeloma, leukemia ora lymphoma.
 52. The method of claim 50, wherein the hematological canceris multiple myeloma.
 53. The method of claim 50, wherein thehematological cancer is non-Hodgkin's lymphoma (NHL) or Hodgkin'slymphoma.
 54. A method for treating a subject with a cancer thatexpresses human B-cell maturation antigen (BCMA), the method comprisingadministering to the subject an effective amount of a compositioncomprising a plurality of antibodies or antibody binding fragmentsthereof that bind to human BCMA, wherein (a) the antibodies or bindingfragments comprise a mature heavy chain variable region and a maturelight chain variable region, wherein the mature heavy chain variableregion complementarity determining regions (CDRs) comprising the aminoacid sequences of SEQ ID NOs:60, 61 and 62, and the mature light chainvariable region comprises CDRs comprising the amino acid sequences ofSEQ ID NOs:90, 91 and 92; and (b) less than 10% of the antibodies orbinding fragments in the composition are fucosylated.
 55. The method ofclaim 54, wherein less than 5% of the antibodies or binding fragments inthe composition are fucosylated.
 56. The method of claim 54, whereinless than 3% of the antibodies or binding fragments in the compositionare fucosylated.
 57. The method of claim 54, wherein less than 2% of theantibodies or binding fragments in the composition are fucosylated. 58.The method of claim 54, wherein the cancer is a hematological cancer.59. The method of claim 58, wherein the hematological cancer is amyeloma, leukemia or a lymphoma.
 60. The method of claim 58, wherein thehematological cancer is multiple myeloma.
 61. The method of claim 58,wherein the hematological cancer is non-Hodgkin's lymphoma (NHL) orHodgkin's lymphoma.
 62. A method for treating a subject with a cancerthat expresses human B-cell maturation antigen (BCMA), the methodcomprising administering to the subject an effective amount of acomposition comprising a plurality of antibodies or binding fragmentsthereof that bind to human BCMA, wherein (a) the antibodies or bindingfragments comprise a mature heavy chain variable region comprising theamino acid sequence of SEQ ID NO:13, and a mature light chain variableregion comprising the amino acid sequence of SEQ ID NO:19; and (b) lessthan 10% of the antibodies or binding fragments in the composition arefucosylated.
 63. The method of claim 62, wherein less than 5% of theantibodies or binding fragments in the composition are fucosylated. 64.The method of claim 62, wherein less than 3% of the antibodies orbinding fragments in the composition are fucosylated.
 65. The method ofclaim 62, wherein less than 2% of the antibodies or binding fragments inthe composition are fucosylated.
 66. The method of claim 62, wherein thecancer is a hematological cancer.
 67. The method of claim 66, whereinthe hematological cancer is a myeloma, leukemia or a lymphoma.
 68. Themethod of claim 66, wherein the hematological cancer is multiplemyeloma.
 69. The method of claim 66, wherein the hematological cancer isnon-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma.
 70. A method fortreating a subject with a cancer that expresses human B-cell maturationantigen (BCMA), the method comprising administering to the subject aneffective amount of a composition comprising a plurality ofimmunoglobulin G1 (IgG1) antibodies that bind to human BCMA, wherein (a)the antibodies comprise a mature heavy chain variable region and amature light chain variable region, wherein the mature heavy chainvariable region comprises complementarity determining regions (CDRs)comprising the amino acid sequences of SEQ ID NOs:60, 61 and 62, and themature light chain variable region comprises CDRs comprising the aminoacid sequences of SEQ ID NOs:90, 91 and 92; and (b) less than 5% of theantibodies in the composition are fucosylated.
 71. The method of claim70, wherein the cancer is a hematological cancer.
 72. The method ofclaim 71, wherein the hematological cancer is a myeloma, leukemia or alymphoma.
 73. The method of claim 71, wherein the hematological canceris multiple myeloma.
 74. The method of claim 71, wherein thehematological cancer is non-Hodgkin's lymphoma (NHL) or Hodgkin'slymphoma.
 75. A method for treating a subject with a cancer thatexpresses human B-cell maturation antigen (BCMA), the method comprisingadministering to the subject an effective amount of a compositioncomprising a plurality of immunoglobulin G1 (IgG1) antibodies that bindto human BCMA, wherein (a) the antibodies comprise (i) a mature heavychain variable region comprising the amino acid sequence of SEQ IDNO:13, and a mature light chain variable region comprising the aminoacid sequence of SEQ ID NO:19; and (ii) a heavy chain constant regioncomprising the amino acid sequence of SEQ ID NO:5, and a light chainconstant region comprising the amino acid sequence of SEQ ID NO:3; and(b) less than 5% of the antibodies in the composition are fucosylated.76. The method of claim 75, wherein the cancer is a hematologicalcancer.
 77. The method of claim 76, wherein the hematological cancer isa myeloma, leukemia or a lymphoma.
 78. The method of claim 76, whereinthe hematological cancer is multiple myeloma.
 79. The method of claim76, wherein the hematological cancer is non-Hodgkin's lymphoma (NHL) orHodgkin's lymphoma.